Method for treating psoriatic arthritis by administering an anti-IL-12 antibody

ABSTRACT

An anti-IL-12 antibody that binds to a portion of the IL-12 protein corresponding to at least one amino acid residue selected from the group consisting of residues 15, 17-21, 23, 40-43, 45-47, 54-56 and 58-62 of the amino acid sequence of the p40 subunit of IL-12, including isolated nucleic acids that encode at least one anti-IL-12 antibody, vectors, host cells, transgenic animals or plants, and methods of making and using thereof have applications in treating IL-12-related disorders, such as psoriatic arthritis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/299,831, filed 18 Nov. 2011, now U.S. Pat. No. 8,329,170, issued 11Dec. 2012, which is a divisional of Ser. No. 11/722,312, filed 8 Dec.2008, now U.S. Pat. No. 8,080,247, issued 20 Dec. 2011, which is anational stage of International Application Number PCT/US2005/046063,filed 20 Dec. 2005, which claims the benefit of U.S. ProvisionalApplication No. 60/637,819, filed 21 Dec. 2004. Parent application Ser.No. 11/722,312 is also a continuation-in-part application of U.S.application Ser. No. 10/975,883, filed 28 Oct. 2004, now U.S. Pat. No.7,279,157, issued 9 Oct. 2007, which is a divisional application of U.S.application Ser. No. 09/920,262, filed 1 Aug. 2001, granted on 7 Jun.2005 under U.S. Pat. No. 6,902,734, which claims priority to U.S.Provisional Application Nos. 60/223,358, filed 7 Aug. 2000, and60/236,827, filed 29 Sep. 2000. The entire contents of each of theaforementioned applications are incorporated herein by reference intheir entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies, including specifiedportions or variants, specific for at least one IL-12 protein orfragment thereof, as well as anti-idiotype antibodies, epitope regionsof the antibodies, portions, variants, and fragments, and nucleic acidsencoding such anti-IL-12 antibodies, complementary nucleic acids,vectors, host cells, and methods of making and using thereof, includingtherapeutic formulations, administration and devices.

BACKGROUND OF THE INVENTION

Interleukin-12 (IL-12) is a heterodimeric cytokine consisting ofglycosylated polypeptide chains of 35 and 40 kD which are disulfidebonded. The cytokine is synthesized and secreted by antigen presentingcells including dendritic cells, monocytes, macrophages, B cells,Langerhans cells and keratinocytes as well as natural killer (NK) cells.IL-12 mediates a variety of biological processes and has been referredto as NK cell stimulatory factor (NKSF), T-cell stimulating factor,cytotoxic T-lymphocyte maturation factor and EBV-transformed B-cell linefactor (Curfs, J. H. A. J., et al., Clinical Microbiology Reviews,10:742-780 (1997)).

Interleukin-12 can bind to the IL-12 receptor expressed on the plasmamembrane of cells (e.g., T cells, NK cell), thereby altering (e.g.,initiating, preventing) biological processes. For example, the bindingof IL-12 to the IL-12 receptor can stimulate the proliferation ofpre-activated T cells and NK cells, enhance the cytolytic activity ofcytotoxic T cells (CTL), NK cells and LAK (lymphokine activated killer)cells, induce production of gamma interferon (IFN GAMMA) by T cells andNK cells and induce differentiation of naive Th0 cells into Th1 cellsthat produce IFN GAMMA and IL-2 (Trinchieri, G., Annual Review ofImmunology, 13:251-276 (1995)). In particular, IL-12 is vital for thegeneration of cytolytic cells (e.g., NK, CTL) and for mounting acellular immune response (e.g., a Th1 cell mediated immune response).Thus, IL-12 is critically important in the generation and regulation ofboth protective immunity (e.g., eradication of infections) andpathological immune responses (e.g., autoimmunity) (Hendrzak, J. A. andBrunda, M. J., Laboratory Investigation, 72:619-637 (1995)).Accordingly, an immune response (e.g., protective or pathogenic) can beenhanced, suppressed or prevented by manipulation of the biologicalactivity of IL-12 in vivo, for example, by means of an antibody.

Non-human mammalian, chimeric, polyclonal (e.g., anti-sera) and/ormonoclonal antibodies (Mabs) and fragments (e.g., proteolytic digestionor fusion protein products thereof) are potential therapeutic agentsthat are being investigated in some cases to attempt to treat certaindiseases. However, such antibodies or fragments can elicit an immuneresponse when administered to humans. Such an immune response can resultin an immune complex-mediated clearance of the antibodies or fragmentsfrom the circulation, and make repeated administration unsuitable fortherapy, thereby reducing the therapeutic benefit to the patient andlimiting the readministration of the antibody or fragment. For example,repeated administration of antibodies or fragments comprising non-humanportions can lead to serum sickness and/or anaphalaxis. In order toavoid these and other problems, a number of approaches have been takento reduce the immunogenicity of such antibodies and portions thereof,including chimerization and humanization, as well known in the art.These and other approaches, however, still can result in antibodies orfragments having some immunogenicity, low affinity, low avidity, or withproblems in cell culture, scale up, production, and/or low yields. Thus,such antibodies or fragments can be less than ideally suited formanufacture or use as therapeutic proteins.

Accordingly, there is a need to provide anti-IL-12 antibodies orfragments that overcome one more of these problems, as well asimprovements over known antibodies or fragments thereof.

SUMMARY OF THE INVENTION

The present invention provides isolated human, anti-IL-12 antibodies,immunoglobulins, fragments, cleavage products and other specifiedportions and variants thereof, as well as anti-IL-12 antibodycompositions, IL-12 anti-idiotype antibodies, epitope regions of theantibodies, portions, variants, and fragments, encoding or complementarynucleic acids, vectors, host cells, compositions, formulations, devices,transgenic animals, transgenic plants, and methods of making and usingthem, as described and enabled herein, in combination with what is knownin the art.

The present invention also provides a method for altering thepotency/activity (e.g., binding) of an antibody by altering one or moreamino acid residues in the antibody variable region and testing theresulting antibody compostion properties.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, or hybridizing to, a polynucleotideencoding specific anti-IL-12 antibodies or anti-idiotype antibodies,comprising at least one specified sequence, domain, portion or variantthereof. The present invention further provides recombinant vectorscomprising said anti-IL-12 antibody nucleic acid molecules, host cellscontaining such nucleic acids and/or recombinant vectors, as well asmethods of making and/or using such antibody nucleic acids, vectorsand/or host cells.

The present invention also provides at least one method for expressingat least one anti-IL-12 antibody, or IL-12 anti-idiotype antibody, in ahost cell, comprising culturing a host cell as described herein underconditions wherein at least one anti-IL-12 antibody is expressed indetectable and/or recoverable amounts.

The present invention also provides at least one composition comprising(a) an isolated anti-IL-12 antibody encoding nucleic acid and/orantibody as described herein; and (b) a suitable and/or pharmaceuticallyacceptable carrier or diluent.

The present invention further provides at least one anti-IL-12 antibodymethod or composition, for administering a therapeutically effectiveamount to modulate or treat at least one IL-12 related condition in acell, tissue, organ, animal or patient and/or, prior to, subsequent to,or during a related condition, as known in the art and/or as describedherein.

The present invention also provides at least one composition, deviceand/or method of delivery of a therapeutically or prophylacticallyeffective amount of at least one anti-IL-12 antibody, according to thepresent invention.

The present invention further provides at least one anti-IL-12 antibodymethod or composition, for diagnosing at least one IL-12 relatedcondition in a cell, tissue, organ, animal or patient and/or, prior to,subsequent to, or during a related condition, as known in the art and/oras described herein.

The present invention also provides at least one composition, deviceand/or method of delivery for diagnosing of at least one anti-IL-12antibody, according to the present invention.

Also provided is a medical device, comprising at least one isolatedmammalian anti-IL-12 antibody of the invention, wherein the device issuitable for contacting or administerting the at least one anti-IL-12antibody, IL-12 anti-idiotypic antibody, nucleic acid molecule,compound, protein, and/or composition.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one isolated mammaliananti-IL-12 antibody of the present invention. The article of manufacturecan optionally have the container as a component of a delivery device orsystem.

The present invention further provides any invention described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the molecular structure of the bound complex of IL-12/p40Fab in a ribbon representation.

FIG. 2 shows the p40 mAb binding site (epitope) represented on themolecular surface in surface and ribbons representations. The D1 domainand Fv are isolated out of the complex structure for clarity. For the D1domain of p40, the molecular surface is shown. The Fv part of the Fab isshown in ribbons. Left panel: view down the antibody binding site, i.e.,the epitope. Middle panel: view ˜90° from view in left panel. Rightpanel: Ribbon representation of the residues of the epitope.

FIG. 3 shows the results of an ELISA evaluation of the IL-12 p40antibody bound to various p40 single mutants.

FIG. 4 shows the relative binding affinity of the p40 mAb to differentp40 muteins.

DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticanti-IL-12 human antibodies and IL-12 anti-idiotype antibodies thereto,as well as compositions and encoding nucleic acid molecules comprisingat least one polynucleotide encoding at least one anti-IL-12 antibody oranti-idiotype antibody. The present invention further includes, but isnot limited to, methods of making and using such nucleic acids andantibodies and anti-idiotype antibodies, including diagnostic andtherapeutic compositions, methods and devices.

As used herein, an “anti-IL-12 antibody,” “IL-12 antibody,” “anti-IL-12antibody portion,” or “anti-IL-12 antibody fragment” and/or “anti-IL-12antibody variant” and the like include any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulinmolecule, such as but not limited to, at least one complementaritydetermining region (CDR) of a heavy or light chain or a ligand bindingportion thereof, a heavy chain or light chain variable region, a heavychain or light chain constant region, a framework region, or any portionthereof, or at least one portion of an IL-12 receptor or bindingprotein, which can be incorporated into an antibody of the presentinvention. Such antibody optionally further affects a specific ligand,such as but not limited to, where such antibody modulates, decreases,increases, antagonizes, angonizes, mitigates, aleviates, blocks,inhibits, abrogates and/or interferes with at least one IL-12 activityor binding, or with IL-12 receptor activity or binding, in vitro, insitu and/or in vivo. As a non-limiting example, a suitable anti-IL-12antibody, specified portion or variant of the present invention can bindat least one IL-12 molecule, or specified portions, variants or domainsthereof. A suitable anti-IL-12 antibody, specified portion, or variantcan also optionally affect at least one of IL-12 activity or function,such as but not limited to, RNA, DNA or protein synthesis, IL-12release, IL-12 receptor signaling, membrane IL-12 cleavage, IL-12activity, IL-12 production and/or synthesis.

The term “antibody” is further intended to encompass antibodies,digestion fragments, specified portions and variants thereof, includingantibody mimetics or comprising portions of antibodies that mimic thestructure and/or function of an antibody or specified fragment orportion thereof, including single chain antibodies and fragmentsthereof. Functional fragments include antigen-binding fragments thatbind to a mammalian IL-12. For example, antibody fragments capable ofbinding to IL-12 or portions thereof, including, but not limited to, Fab(e.g., by papain digestion), Fab′ (e.g., by pepsin digestion and partialreduction) and F(ab′)₂ (e.g., by pepsin digestion), facb (e.g., byplasmin digestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd(e.g., by pepsin digestion, partial reduction and reaggregation), Fv orscFv (e.g., by molecular biology techniques) fragments, are encompassedby the invention (see, e.g., Colligan, Immunology, supra).

Such fragments can be produced by enzymatic cleavage, synthetic orrecombinant techniques, as known in the art and/or as described herein.Antibodies can also be produced in a variety of truncated forms usingantibody genes in which one or more stop codons have been introducedupstream of the natural stop site. For example, a combination geneencoding a F(ab′)₂ heavy chain portion can be designed to include DNAsequences encoding the C_(H)1 domain and/or hinge region of the heavychain. The various portions of antibodies can be joined togetherchemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques.

As used herein, the term “human antibody” refers to an antibody in whichsubstantially every part of the protein (e.g., CDR, framework, C_(L),C_(H) domains (e.g., C_(H)1, C_(H)2, C_(H)3), hinge, (V_(L), V_(H))) issubstantially non-immunogenic in humans, with only minor sequencechanges or variations. A “human antibody” may also be an antibody thatis derived from or closely matches human germline immunoglobulinsequences. Human antibodies may include amino acid residues not encodedby germline immunoglobulin sequences (e.g., mutations introduced byrandom or site-specific mutagenesis in vitro or by somatic mutation invivo). Often, this means that the human antibody is substantiallynon-immunogenic in humans. Human antibodies have been classified intogroupings based on their amino acid sequence similarities. Accordingly,using a sequence similarity search, an antibody with a similar linearsequence can be chosen as a template to create a human antibody.Similarly, antibodies designated primate (monkey, baboon, chimpanzee,etc.); rodent (mouse, rat, rabbit, guinea pig, hamster, and the like)and other mammals designate such species, sub-genus, genus, sub-family,and family specific antibodies. Further, chimeric antibodies can includeany combination of the above. Such changes or variations optionally andpreferably retain or reduce the immunogenicity in humans or otherspecies relative to non-modified antibodies. Thus, a human antibody isdistinct from a chimeric or humanized antibody.

It is pointed out that a human antibody can be produced by a non-humananimal or prokaryotic or eukaryotic cell that is capable of expressingfunctionally rearranged human immunoglobulin (e.g., heavy chain and/orlight chain) genes. Further, when a human antibody is a single chainantibody, it can comprise a linker peptide that is not found in nativehuman antibodies. For example, an Fv can comprise a linker peptide, suchas two to about eight glycine or other amino acid residues, whichconnects the variable region of the heavy chain and the variable regionof the light chain. Such linker peptides are considered to be of humanorigin.

Bispecific, heterospecific, heteroconjugate or similar antibodies canalso be used that are monoclonal, preferably, human or humanized,antibodies that have binding specificities for at least two differentantigens. In the present case, one of the binding specificities is forat least one IL-12 protein, the other one is for any other antigen.Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy chain-light chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, Nature 305:537 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. The purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed, e.g., in WO 93/08829, U.S. Pat. Nos.6,210,668, 6,193,967, 6,132,992, 6,106,833, 6,060,285, 6,037,453,6,010,902, 5,989,530, 5,959,084, 5,959,083, 5,932,448, 5,833,985,5,821,333, 5,807,706, 5,643,759, 5,601,819, 5,582,996, 5,496,549,4,676,980, WO 91/00360, WO 92/00373, EP 03089, Traunecker et al., EMBOJ. 10:3655 (1991), Suresh et al., Methods in Enzymology 121:210 (1986),each entirely incorporated herein by reference.

Anti-IL-12 antibodies (also termed IL-12 antibodies) useful in themethods and compositions of the present invention can optionally becharacterized by high affinity binding to IL-12 and, optionally andpreferably, having low toxicity. In particular, an antibody, specifiedfragment or variant of the invention, where the individual components,such as the variable region, constant region and framework, individuallyand/or collectively, optionally and preferably possess lowimmunogenicity, is useful in the present invention. The antibodies thatcan be used in the invention are optionally characterized by theirability to treat patients for extended periods with measurablealleviation of symptoms and low and/or acceptable toxicity. Low oracceptable immunogenicity and/or high affinity, as well as othersuitable properties, can contribute to the therapeutic results achieved.“Low immunogenicity” is defined herein as raising significant HAHA, HACAor HAMA responses in less than about 75%, or preferably less than about50% of the patients treated and/or raising low titres in the patienttreated (less than about 300, preferably less than about 100 measuredwith a double antigen enzyme immunoassay) (Elliott et al., Lancet344:1125-1127 (1994), entirely incorporated herein by reference). “Lowimmunogenicity” can also be defined as the incidence of titrable levelsof antibodies to the anti-IL-12 antibody in patients treated withanti-IL-12 antibody as occurring in less than 25% of patients treated,preferably, in less than 10% of patients treated with the recommendeddose for the recommended course of therapy during the treatment period.

Utility

The isolated nucleic acids of the present invention can be used forproduction of at least one anti-IL-12 antibody or specified variantthereof, which can be used to measure or effect in an cell, tissue,organ or animal (including mammals and humans), to diagnose, monitor,modulate, treat, alleviate, help prevent the incidence of, or reduce thesymptoms of, at least one IL-12 condition, selected from, but notlimited to, at least one of an immune disorder or disease, acardiovascular disorder or disease, an infectious, malignant, and/orneurologic disorder or disease, or other known or specified IL-12related condition.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment, alleviation, prevention, or reduction insymptoms, effects or mechanisms. The effective amount can comprise anamount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple orcontinuous administration, or to achieve a serum concentration of0.01-5000 μg/ml serum concentration per single, multiple, or continuousadministration, or any effective range or value therein, as done anddetermined using known methods, as described herein or known in therelevant arts.

Citations

All publications or patents cited herein, whether or not specificallydesignated, are entirely incorporated herein by reference as they showthe state of the art at the time of the present invention and/or toprovide description and enablement of the present invention.Publications refer to any scientific or patent publications, or anyother information available in any media format, including all recorded,electronic or printed formats. The following references are entirelyincorporated herein by reference: Ausubel, et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y.(1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual,2^(nd) Edition, Cold Spring Harbor, N.Y. (1989); Harlow and Lane,antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y. (1989);Colligan, et al., eds., Current Protocols in Immunology, John Wiley &Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001).

Antibodies of the Present Invention—Production and Generation

At least one anti-IL-12 antibody of the present invention can beoptionally produced by a cell line, a mixed cell line, an immortalizedcell or clonal population of immortalized cells, as well known in theart. See, e.g., Ausubel, et al., ed., Current Protocols in MolecularBiology, John Wiley & Sons, Inc., NY, N.Y. (1987-2001); Sambrook, etal., Molecular Cloning: A Laboratory Manual, 2^(nd) Edition, Cold SpringHarbor, N.Y. (1989); Harlow and Lane, antibodies, a Laboratory Manual,Cold Spring Harbor, N.Y. (1989); Colligan, et al., eds., CurrentProtocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001);Colligan et al., Current Protocols in Protein Science, John Wiley &Sons, NY, N.Y., (1997-2001), each entirely incorporated herein byreference.

Human antibodies that are specific for human IL-12 proteins or fragmentsthereof can be raised against an appropriate immunogenic antigen, suchas an isolated IL-12 protein and/or a portion thereof (includingsynthetic molecules, such as synthetic peptides). Other specific orgeneral mammalian antibodies can be similarly raised. Preparation ofimmunogenic antigens and monoclonal antibody production can be performedusing any suitable technique.

In one approach, a hybridoma is produced by fusing a suitable immortalcell line (e.g., a myeloma cell line, such as, but not limited to,Sp2/0, Sp2/0-AG14, NSO, NS1, NS2, AE-1, L.5, L243, P3X63Ag8.653, Sp2SA3, Sp2 MAI, Sp2 SS1, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-1,JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMALWA, NEURO2A, or the like, or heteromylomas, fusion products thereof, or any cellor fusion cell derived therefrom, or any other suitable cell line asknown in the art) (see, e.g., www.atcc.org, www.lifetech.com., and thelike), with antibody producing cells, such as, but not limited to,isolated or cloned spleen, peripheral blood, lymph, tonsil, or otherimmune or B cell containing cells, or any other cells expressing heavyor light chain constant or variable or framework or CDR sequences,either as endogenous or heterologous nucleic acid, as recombinant orendogenous, viral, bacterial, algal, prokaryotic, amphibian, insect,reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate,eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA,chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triplestranded, hybridized, and the like or any combination thereof. See,e.g., Ausubel, supra, and Colligan, Immunology, supra, chapter 2,entirely incorporated herein by reference.

Antibody producing cells can also be obtained from the peripheral bloodor, preferably, the spleen or lymph nodes, of humans or other suitableanimals that have been immunized with the antigen of interest. Any othersuitable host cell can also be used for expressing heterologous orendogenous nucleic acid encoding an antibody, specified fragment orvariant thereof, of the present invention. The fused cells (hybridomas)or recombinant cells can be isolated using selective culture conditionsor other suitable known methods, and cloned by limiting dilution or cellsorting, or other known methods. Cells which produce antibodies with thedesired specificity can be selected by a suitable assay (e.g., ELISA).

Other suitable methods of producing or isolating antibodies of therequisite specificity can be used, including, but not limited to,methods that select recombinant antibody from a peptide or proteinlibrary (e.g., but not limited to, a bacteriophage, ribosome,oligonucleotide, RNA, cDNA, or the like, display library; e.g., asavailable from Cambridge antibody Technologies, Cambridgeshire, UK;MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK;Bioinvent, Lund, Sweden; Dyax Corp., Enzon, Affymax/Biosite; Xoma,Berkeley, Calif.; Ixsys. See, e.g., EP 368,684, PCT/GB91/01134;PCT/GB92/01755; PCT/GB92/002240; PCT/GB92/00883; PCT/GB93/00605; U.S.Ser. No. 08/350,260 (May 12, 1994); PCT/GB94/01422; PCT/GB94/02662;PCT/GB97/01835; (CAT/MRC); WO90/14443; WO90/14424; WO90/14430;PCT/US94/1234; WO92/18619; WO96/07754; (Scripps); WO96/13583, WO97/08320(MorphoSys); WO95/16027 (BioInvent); WO88/06630; WO90/3809 (Dyax); U.S.Pat. No. 4,704,692 (Enzon); PCT/US91/02989 (Affymax); WO89/06283; EP 371998; EP 550 400; (Xoma); EP 229 046; PCT/US91/07149 (Ixsys); orstochastically generated peptides or proteins—U.S. Pat. Nos. 5,723,323,5,763,192, 5,814,476, 5,817,483, 5,824,514, 5,976,862, WO 86/05803, EP590 689 (Ixsys, predecessor of Applied Molecular Evolution (AME), eachentirely incorporated herein by reference)) or that rely uponimmunization of transgenic animals (e.g., SCID mice, Nguyen et al.,Microbiol. Immunol. 41:901-907 (1997); Sandhu et al., Crit. Rev.Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93:154-161 (1998),each entirely incorporated by reference as well as related patents andapplications) that are capable of producing a repertoire of humanantibodies, as known in the art and/or as described herein. Suchtechniques, include, but are not limited to, ribosome display (Hanes etal., Proc. Natl. Acad. Sci. USA, 94:4937-4942 (May 1997); Hanes et al.,Proc. Natl. Acad. Sci. USA, 95:14130-14135 (November 1998)); single cellantibody producing technologies (e.g., selected lymphocyte antibodymethod (“SLAM”) (U.S. Pat. No. 5,627,052, Wen et al., J. Immunol.17:887-892 (1987); Babcook et al., Proc. Natl. Acad. Sci. USA93:7843-7848 (1996)); gel microdroplet and flow cytometry (Powell etal., Biotechnol. 8:333-337 (1990); One Cell Systems, Cambridge, Mass.;Gray et al., J. Imm. Meth. 182:155-163 (1995); Kenny et al.,Bio/Technol. 13:787-790 (1995)); B-cell selection (Steenbakkers et al.,Molec. Biol. Reports 19:125-134 (1994); Jonak et al., Progress Biotech,Vol. 5, In Vitro Immunization in Hybridoma Technology, Borrebaeck, ed.,Elsevier Science Publishers B.V., Amsterdam, Netherlands (1988)).

Methods for engineering or humanizing non-human or human antibodies canalso be used and are well known in the art. Generally, a humanized orengineered antibody has one or more amino acid residues from a sourcethat is non-human, e.g., but not limited to, mouse, rat, rabbit,non-human primate or other mammal. These non-human amino acid residuesare replaced by residues often referred to as “import” residues, whichare typically taken from an “import” variable, constant or other domainof a known human sequence.

Known human Ig sequences are disclosed, e.g.,www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast;www.atcc.org/phage/hdb.html; www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php;www.kabatdatabase.com/top.html; ftp.ncbi.nih.gov/repository/kabat;www.sciquest.com; www.abcam.com;www.antibodyresource.com/onlinecomp.html;www.public.iastate.edu/˜pedro/research_tools.html;www.whfreeman.com/immunology/CH05/kuby05.htm;www.hhmi.org/grants/lectures/1996/vlab;www.path.cam.ac.uk/˜mrc7/mikeimages.html;mcb.harvard.edu/BioLinks/Immunology.html; www.immunologylink.com;pathbox.wust1.edu/˜hcenter/index.html; www.appliedbiosystems.com;www.nal.usda.gov/awic/pubs/antibody;www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com;www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org;baserv.uci.kun.n1/˜jraats/links1.html;www.recab.uni-hd.de/immuno.bme.nwu.edu; www.mrc-cpe.cam.ac.uk;www.ibt.unam.mx/vir/V_mice.html; http://www.bioinforg.uk/abs;antibody.bath.ac.uk; www.unizh.ch; www.cryst.bbk.ac.uk/˜ubcg07s;www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html;www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html;www.ibt.unam.mx/vir/structure/stat_aim.html;www.biosci.missouri.edu/smithgp/index.html; www.jerini.de; Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference.

Such imported sequences can be used to reduce immunogenicity or reduce,enhance or modify binding, affinity, on-rate, off-rate, avidity,specificity, half-life, or any other suitable characteristic, as knownin the art. In general, the CDR residues are directly and mostsubstantially involved in influencing antigen binding. Accordingly, partor all of the non-human or human CDR sequences are maintained while thenon-human sequences of the variable and constant regions may be replacedwith human or other amino acids.

Antibodies can also optionally be humanized or human antibodiesengineered with retention of high affinity for the antigen and otherfavorable biological properties. To achieve this goal, humanized (orhuman) antibodies can be optionally prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, framework (FR) residuescan be selected and combined from the consensus and import sequences sothat the desired antibody characteristic, such as increased affinity forthe target antigen(s), is achieved.

In addition, the human IL-12 antibody of the present invention maycomprise a human germline light chain framework. In particularembodiments, the light chain germline sequence is selected from human VKsequences including, but not limited to, A1, A10, A11, A14, A17, A18,A19, A2, A20, A23, A26, A27, A3, A30, A5, A7, B2, B3, L1, L10, L11, L12,L14, L15, L16, L18, L19, L2, L20, L22, L23, L24, L25, L4/18a, L5, L6,L8, L9, O1, O1, O12, O14, O18, O2, O4, and O8. In certain embodiments,this light chain human germline framework is selected from V1-11, V1-13,V1-16, V1-17, V1-18, V1-19, V1-2, V1-20, V1-22, V1-3, V1-4, V1-5, V1-7,V1-9, V2-1, V2-11, V2-13, V2-14, V2-15, V2-17, V2-19, V2-6, V2-7, V2-8,V3-2, V3-3, V3-4, V4-1, V4-2, V4-3, V4-4, V4-6, V5-1, V5-2, V5-4, andV5-6.

In other embodiments, the human IL-12 antibody of the present inventionmay comprise a human germline heavy chain framework. In particularembodiments, this heavy chain human germline framework is selected fromVH1-18, VH1-2, VH1-24, VH1-3, VH1-45, VH1-46, VH1-58, VH1-69, VH1-8,VH2-26, VH2-5, VH2-70, VH3-11, VH3-13, VH3-15, VH3-16, VH3-20, VH3-21,VH3-23, VH3-30, VH3-33, VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53,VH3-64, VH3-66, VH3-7, VH3-72, VH3-73, VH3-74, VH3-9, VH4-28, VH4-31,VH4-34, VH4-39, VH4-4, VH4-59, VH4-61, VH5-51, VH6-1, and VH7-81.

In particular embodiments, the light chain variable region and/or heavychain variable region comprises a framework region or at least a portionof a framework region (e.g., containing 2 or 3 subregions, such as FR2and FR3). In certain embodiments, at least FRL1, FRL2, FRL3, or FRL4 isfully human. In other embodiments, at least FRH1, FRH2, FRH3, or FRH4 isfully human. In some embodiments, at least FRL1, FRL2, FRL3, or FRL4 isa germline sequence (e.g., human germline) or comprises human consensussequences for the particular framework (readily available at the sourcesof known human Ig sequences described above). In other embodiments, atleast FRH1, FRH2, FRH3, or FRH4 is a germline sequence (e.g., humangermline) or comprises human consensus sequences for the particularframework. In preferred embodiments, the framework region is a fullyhuman framework region.

Humanization or engineering of antibodies of the present invention canbe performed using any known method, such as but not limited to thosedescribed in, Winter (Jones et al., Nature 321:522 (1986); Riechmann etal., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988)),Sims et al., J. Immunol. 151: 2296 (1993); Chothia and Lesk, J. Mol.Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A.89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), U.S. Pat.Nos. 5,723,323, 5,976,862, 5,824,514, 5,817,483, 5,814,476, 5,763,192,5,723,323, 5,766,886, 5,714,352, 6,204,023, 6,180,370, 5,693,762,5,530,101, 5,585,089, 5,225,539; 4,816,567, PCT/: US98/16280,US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134,GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, each entirelyincorporated herein by reference, included references cited therein.

In certain embodiments, the antibody comprises an altered (e.g.,mutated) Fc region. For example, in some embodiments, the Fc region hasbeen altered to reduce or enhance the effector functions of theantibody. In some embodiments, the Fc region is an isotype selected fromIgM, IgA, IgG, IgE, or other isotype. Alternatively or additionally, itmay be useful to combine amino acid modifications with one or morefurther amino acid modifications that alter C1q binding and/or thecomplement dependent cytotoxicity function of the Fc region of an IL-12binding molecule. The starting polypeptide of particular interest may beone that binds to C1q and displays complement dependent cytotoxicity(CDC). Polypeptides with pre-existing C1q binding activity, optionallyfurther having the ability to mediate CDC may be modified such that oneor both of these activities are enhanced Amino acid modifications thatalter C1q and/or modify its complement dependent cytotoxicity functionare described, for example, in WO0042072, which is hereby incorporatedby reference.

As disclosed above, one can design an Fc region of the human IL-12antibody of the present invention with altered effector function, e.g.,by modifying C1q binding and/or FcγR binding and thereby changingcomplement dependent cytotoxicity (CDC) activity and/orantibody-dependent cell-mediated cytotoxicity (ADCC) activity. “Effectorfunctions” are responsible for activating or diminishing a biologicalactivity (e.g., in a subject). Examples of effector functions include,but are not limited to: C1q binding; CDC; Fc receptor binding; ADCC;phagocytosis; down regulation of cell surface receptors (e.g., B cellreceptor; BCR), etc. Such effector functions may require the Fc regionto be combined with a binding domain (e.g., an antibody variable domain)and can be assessed using various assays (e.g., Fc binding assays, ADCCassays, CDC assays, etc.).

For example, one can generate a variant Fc region of the human IL-12antibody with improved C1q binding and improved FcγRIII binding (e.g.,having both improved ADCC activity and improved CDC activity).Alternatively, if it is desired that effector function be reduced orablated, a variant Fc region can be engineered with reduced CDC activityand/or reduced ADCC activity. In other embodiments, only one of theseactivities may be increased, and, optionally, also the other activityreduced (e.g., to generate an Fc region variant with improved ADCCactivity, but reduced CDC activity and vice versa).

Fc mutations can also be introduced in engineer to alter theirinteraction with the neonatal Fc receptor (FcRn) and improve theirpharmacokinetic properties. A collection of human Fc variants withimproved binding to the FcRn have been described (Shields et al.,(2001). High resolution mapping of the binding site on human IgG1 forFcγRI, FcγRII, FcγRIII, and FcRn and design of IgG1 variants withimproved binding to the FcγR, J. Biol. Chem. 276:6591-6604).

Another type of amino acid substitution serves to alter theglycosylation pattern of the Fc region of the human IL-12 antibody.Glycosylation of an Fc region is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. O-linked glycosylation refers to theattachment of one of the sugars N-aceylgalactosamine, galactose, orxylose to a hydroxyamino acid, most commonly serine or threonine,although 5-hydroxyproline or 5-hydroxylysine may also be used. Therecognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain peptide sequences areasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline. Thus, the presence of either of these peptidesequences in a polypeptide creates a potential glycosylation site.

The glycosylation pattern may be altered, for example, by deleting oneor more glycosylation site(s) found in the polypeptide, and/or addingone or more glycosylation sites that are not present in the polypeptide.Addition of glycosylation sites to the Fc region of a human IL-12antibody is conveniently accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). An exemplaryglycosylation variant has an amino acid substitution of residue Asn 297of the heavy chain. The alteration may also be made by the addition of,or substitution by, one or more serine or threonine residues to thesequence of the original polypeptide (for O-linked glycosylation sites).Additionally, a change of Asn 297 to Ala can remove one of theglycosylation sites.

In certain embodiments, the human IL-12 antibody of the presentinvention is expressed in cells that express beta(1,4)-N-acetylglucosaminyltransferase III (GnT III), such that GnT IIIadds GlcNAc to the human IL-12 antibody. Methods for producingantibodies in such a fashion are provided in WO/9954342, WO/03011878,patent publication 20030003097A1, and Umana et al., NatureBiotechnology, 17:176-180, February 1999; all of which are hereinspecifically incorporated by reference in their entireties.

The anti-IL-12 antibody can also be optionally generated by immunizationof a transgenic animal (e.g., mouse, rat, hamster, non-human primate,and the like) capable of producing a repertoire of human antibodies, asdescribed herein and/or as known in the art. Cells that produce a humananti-IL-12 antibody can be isolated from such animals and immortalizedusing suitable methods, such as the methods described herein.

Transgenic mice that can produce a repertoire of human antibodies thatbind to human antigens can be produced by known methods (e.g., but notlimited to, U.S. Pat. Nos. 5,770,428, 5,569,825, 5,545,806, 5,625,126,5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al.;Jakobovits et al. WO 98/50433, Jakobovits et al. WO 98/24893, Lonberg etal. WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585,Kucherlapate et al. WO 96/34096, Kucherlapate et al. EP 0463 151 B1,Kucherlapate et al. EP 0710 719 A1, Surani et al. U.S. Pat. No.5,545,807, Bruggemann et al. WO 90/04036, Bruggemann et al. EP 0438 474B1, Lonberg et al. EP 0814 259 A2, Lonberg et al. GB 2 272 440 A,Lonberg et al. Nature 368:856-859 (1994), Taylor et al., Int. Immunol.6(4)579-591 (1994), Green et al, Nature Genetics 7:13-21 (1994), Mendezet al., Nature Genetics 15:146-156 (1997), Taylor et al., Nucleic AcidsResearch 20(23):6287-6295 (1992), Tuaillon et al., Proc Natl Acad SciUSA 90(8)3720-3724 (1993), Lonberg et al., Int Rev Immunol 13(1):65-93(1995) and Fishwald et al., Nat Biotechnol 14(7):845-851 (1996), whichare each entirely incorporated herein by reference). Generally, thesemice comprise at least one transgene comprising DNA from at least onehuman immunoglobulin locus that is functionally rearranged, or which canundergo functional rearrangement. The endogenous immunoglobulin loci insuch mice can be disrupted or deleted to eliminate the capacity of theanimal to produce antibodies encoded by endogenous genes.

Screening antibodies for specific binding to similar proteins orfragments can be conveniently achieved using peptide display libraries.This method involves the screening of large collections of peptides forindividual members having the desired function or structure. Antibodyscreening of peptide display libraries is well known in the art. Thedisplayed peptide sequences can be from 3 to 5000 or more amino acids inlength, frequently from 5-100 amino acids long, and often from about 8to 25 amino acids long. In addition to direct chemical synthetic methodsfor generating peptide libraries, several recombinant DNA methods havebeen described. One type involves the display of a peptide sequence onthe surface of a bacteriophage or cell. Each bacteriophage or cellcontains the nucleotide sequence encoding the particular displayedpeptide sequence. Such methods are described in PCT Patent PublicationNos. 91/17271, 91/18980, 91/19818, and 93/08278.

Other systems for generating libraries of peptides have aspects of bothin vitro chemical synthesis and recombinant methods. See, PCT PatentPublication Nos. 92/05258, 92/14843, and 96/19256. See also, U.S. Pat.Nos. 5,658,754; and 5,643,768. Peptide display libraries, vector, andscreening kits are commercially available from such suppliers asInvitrogen (Carlsbad, Calif.), and Cambridge antibody Technologies(Cambridgeshire, UK). See, e.g., U.S. Pat. Nos. 4,704,692, 4,939,666,4,946,778, 5,260,203, 5,455,030, 5,518,889, 5,534,621, 5,656,730,5,763,733, 5,767,260, 5,856,456, assigned to Enzon; U.S. Pat. Nos.5,223,409, 5,403,484, 5,571,698, 5,837,500, assigned to Dyax, U.S. Pat.Nos. 5,427,908, 5,580,717, assigned to Affymax; U.S. Pat. No. 5,885,793,assigned to Cambridge antibody Technologies; U.S. Pat. No. 5,750,373,assigned to Genentech, U.S. Pat. Nos. 5,618,920, 5,595,898, 5,576,195,5,698,435, 5,693,493, 5,698,417, assigned to Xoma, Colligan, supra;Ausubel, supra; or Sambrook, supra, each of the above patents andpublications entirely incorporated herein by reference.

Antibodies of the present invention can also be prepared using at leastone anti-IL-12 antibody encoding nucleic acid to provide transgenicanimals or mammals, such as goats, cows, horses, sheep, rabbits, and thelike, that produce such antibodies in their milk. Such animals can beprovided using known methods. See, e.g., but not limited to, U.S. Pat.Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362;5,304,489, and the like, each of which is entirely incorporated hereinby reference.

Antibodies of the present invention can additionally be prepared usingat least one anti-IL-12 antibody encoding nucleic acid to providetransgenic plants and cultured plant cells (e.g., but not limited to,tobacco and maize) that produce such antibodies, specified portions orvariants in the plant parts or in cells cultured therefrom. As anon-limiting example, transgenic tobacco leaves expressing recombinantproteins have been successfully used to provide large amounts ofrecombinant proteins, e.g., using an inducible promoter. See, e.g.,Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize have been used toexpress mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainantibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencescited therein. Thus, antibodies of the present invention can also beproduced using transgenic plants, according to known methods. See also,e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (October,1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., PlantPhysiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans.22:940-944 (1994); and references cited therein. Each of the abovereferences is entirely incorporated herein by reference.

The antibodies of the invention can bind human IL-12 with a wide rangeof affinities (K_(D)). In a preferred embodiment, at least one human mAbof the present invention can optionally bind human IL-12 with highaffinity. For example, a human mAb can bind human IL-12 with a K_(D)equal to or less than about 10⁻⁷ M, such as but not limited to, 0.1-9.9(or any range or value therein)×10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²,10⁻¹³ or any range or value therein.

The affinity or avidity of an antibody for an antigen can be determinedexperimentally using any suitable method. (See, for example, Berzofsky,et al., “Antibody-Antigen Interactions,” In Fundamental Immunology,Paul, W. E., Ed., Raven Press: New York, N.Y. (1984); Kuby, JanisImmunology, W. H. Freeman and Company: New York, N.Y. (1992); andmethods described herein). The measured affinity of a particularantibody-antigen interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other antigen-binding parameters (e.g., K_(D), K_(a),K_(d)) are preferably made with standardized solutions of antibody andantigen, and a standardized buffer, such as the buffer described herein.

Nucleic Acid Molecules

Using the information provided herein, for example, the nucleotidesequences encoding at least 70-100% of the contiguous amino acids of atleast one of the light or heavy chain variable or CDR regions of SEQ IDNOS: 1, 2, 3, 4, 5, 6, 7, 8, among other sequences disclosed herein,specified fragments, variants or consensus sequences thereof, or adeposited vector comprising at least one of these sequences, a nucleicacid molecule of the present invention encoding at least one anti-IL-12antibody can be obtained using methods described herein or as known inthe art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combinations thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally, with one or more introns, e.g., but not limited to, at leastone specified portion of at least one CDR, such as CDR1, CDR2 and/orCDR3 of at least one heavy chain (e.g., SEQ ID NOS:1-3) or light chain(e.g., SEQ ID NOS:4-6); nucleic acid molecules comprising the codingsequence for an anti-IL-12 antibody or variable region (e.g., light andheavy chain variable regions of SEQ ID NOS:7 and 8); and nucleic acidmolecules which comprise a nucleotide sequence substantially differentfrom those described above but which, due to the degeneracy of thegenetic code, still encode at least one anti-IL-12 antibody as describedherein and/or as known in the art. Of course, the genetic code is wellknown in the art. Thus, it would be routine for one skilled in the artto generate such degenerate nucleic acid variants that code for specificanti-IL-12 antibodies of the present invention. See, e.g., Ausubel, etal., supra, and such nucleic acid variants are included in the presentinvention. Non-limiting examples of isolated nucleic acid molecules ofthe present invention include nucleic acids encoding HC CDR1, HC CDR2,HC CDR3, LC CDR1, LC CDR2, and LC CDR3, respectively.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an anti-IL-12 antibody caninclude, but are not limited to, those encoding the amino acid sequenceof an antibody fragment, by itself; the coding sequence for the entireantibody or a portion thereof; the coding sequence for an antibody,fragment or portion, as well as additional sequences, such as the codingsequence of at least one signal leader or fusion peptide, with orwithout the aforementioned additional coding sequences, such as at leastone intron, together with additional, non-coding sequences, includingbut not limited to, non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription,mRNA processing, including splicing and polyadenylation signals (forexample, ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding anantibody can be fused to a marker sequence, such as a sequence encodinga peptide that facilitates purification of the fused antibody comprisingan antibody fragment or portion.

Polynucleotides Selectively Hybridizing to a Polynucleotide as DescribedHerein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising suchpolynucleotides. For example, polynucleotides of the present inventioncan be used to identify, isolate, or amplify partial or full-lengthclones in a deposited library. In some embodiments, the polynucleotidesare genomic or cDNA sequences isolated, or otherwise complementary to, acDNA from a human or mammalian nucleic acid library.

Preferably, the cDNA library comprises at least 80% full-lengthsequences, preferably, at least 85% or 90% full-length sequences, and,more preferably, at least 95% full-length sequences. The cDNA librariescan be normalized to increase the representation of rare sequences. Lowor moderate stringency hybridization conditions are typically, but notexclusively, employed with sequences having a reduced sequence identityrelative to complementary sequences. Moderate and high stringencyconditions can optionally be employed for sequences of greater identity.Low stringency conditions allow selective hybridization of sequenceshaving about 70% sequence identity and can be employed to identifyorthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an antibody encoded by the polynucleotides described herein.The polynucleotides of this invention embrace nucleic acid sequencesthat can be employed for selective hybridization to a polynucleotideencoding an antibody of the present invention. See, e.g., Ausubel,supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, and/or (d) combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention, excluding the coding sequence, is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. (See, e.g.,Ausubel, supra; or Sambrook, supra)

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, are well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra)

Nucleic Acid Screening and Isolation Methods

A cDNA or genomic library can be screened using a probe based upon thesequence of a polynucleotide of the present invention, such as thosedisclosed herein. Probes can be used to hybridize with genomic DNA orcDNA sequences to isolate homologous genes in the same or differentorganisms. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled byone or more of temperature, ionic strength, pH and the presence of apartially denaturing solvent, such as formamide. For example, thestringency of hybridization is conveniently varied by changing thepolarity of the reactant solution through, for example, manipulation ofthe concentration of formamide within the range of 0% to 50%. The degreeof complementarity (sequence identity) required for detectable bindingwill vary in accordance with the stringency of the hybridization mediumand/or wash medium. The degree of complementarity will optimally be100%, or 70-100%, or any range or value therein. However, it should beunderstood that minor sequence variations in the probes and primers canbe compensated for by reducing the stringency of the hybridizationand/or wash medium.

Methods of amplification of RNA or DNA are well known in the art and canbe used according to the present invention without undueexperimentation, based on the teaching and guidance presented herein.

Known methods of DNA or RNA amplification include, but are not limitedto, polymerase chain reaction (PCR) and related amplification processes(see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, 4,965,188,to Mullis, et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 to Tabor, etal; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464 to Wilson,et al.; U.S. Pat. No. 5,091,310 to Innis; U.S. Pat. No. 5,066,584 toGyllensten, et al; U.S. Pat. No. 4,889,818 to Gelfand, et al; U.S. Pat.No. 4,994,370 to Silver, et al; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold) and RNA mediated amplification that usesanti-sense RNA to the target sequence as a template for double-strandedDNA synthesis (U.S. Pat. No. 5,130,238 to Malek, et al, with thetradename NASBA), the entire contents of which references areincorporated herein by reference. (See, e.g., Ausubel, supra; orSambrook, supra.)

For instance, polymerase chain reaction (PCR) technology can be used toamplify the sequences of polynucleotides of the present invention andrelated genes directly from genomic DNA or cDNA libraries. PCR and otherin vitro amplification methods can also be useful, for example, to clonenucleic acid sequences that code for proteins to be expressed, to makenucleic acids to use as probes for detecting the presence of the desiredmRNA in samples, for nucleic acid sequencing, or for other purposes.Examples of techniques sufficient to direct persons of skill through invitro amplification methods are found in Berger, supra, Sambrook, supra,and Ausubel, supra, as well as Mullis, et al., U.S. Pat. No. 4,683,202(1987); and Innis, et al., PCR Protocols A Guide to Methods andApplications, Eds., Academic Press Inc., San Diego, Calif. (1990).Commercially available kits for genomic PCR amplification are known inthe art. See, e.g., Advantage-GC Genomic PCR Kit (Clontech).Additionally, e.g., the T4 gene 32 protein (Boehringer Mannheim) can beused to improve yield of long PCR products.

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example, a cDNA or a genomicsequence encoding an antibody of the present invention, can be used toconstruct a recombinant expression cassette that can be introduced intoat least one desired host cell. A recombinant expression cassette willtypically comprise a polynucleotide of the present invention operablylinked to transcriptional initiation regulatory sequences that willdirect the transcription of the polynucleotide in the intended hostcell. Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in the intron) of a non-heterologousform of a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution.

Vectors and Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one anti-IL-12 antibody by recombinant techniques, as iswell known in the art. See, e.g., Sambrook, et al., supra; Ausubel, etal., supra, each entirely incorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced in a precipitate, such as a calcium phosphate precipitate,or in a complex with a charged lipid. If the vector is a virus, it canbe packaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites for transcriptioninitiation, termination and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will preferably include atranslation initiating at the beginning and a termination codon (e.g.,UAA, UGA or UAG) appropriately positioned at the end of the mRNA to betranslated, with UAA and UAG preferred for mammalian or eukaryotic cellexpression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but are not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one antibody of the present invention can be expressed in amodified form, such as a fusion protein, and can include not onlysecretion signals, but also additional heterologous functional regions.For instance, a region of additional amino acids, particularly chargedamino acids, can be added to the N-terminus of an antibody to improvestability and persistence in the host cell, during purification, orduring subsequent handling and storage. Also, peptide moieties can beadded to an antibody of the present invention to facilitatepurification. Such regions can be removed prior to final preparation ofan antibody or at least one fragment thereof. Such methods are describedin many standard laboratory manuals, such as Sambrook, supra, Chapters17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention. Alternatively, nucleic acids of thepresent invention can be expressed in a host cell by turning on (bymanipulation) in a host cell that contains endogenous DNA encoding anantibody of the present invention. Such methods are well known in theart, e.g., as described in U.S. Pat. Nos. 5,580,734, 5,641,670,5,733,746, and 5,733,761, entirely incorporated herein by reference.

Illustrative of cell cultures useful for the production of theantibodies, specified portions or variants thereof, are mammalian cells.Mammalian cell systems often will be in the form of monolayers of cellsalthough mammalian cell suspensions or bioreactors can also be used. Anumber of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCCCRL-26) cell lines, Cos-7 cells, CHO cells, hep G2 cells, P3X63Ag8.653,SP2/0-Ag14, 293 cells, HeLa cells and the like, which are readilyavailable from, for example, American Type Culture Collection, Manassas,Va. (www.atcc.org). Preferred host cells include cells of lymphoidorigin, such as myeloma and lymphoma cells. Particularly preferred hostcells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) andSP2/0-Ag14 cells (ATCC Accession Number CRL-1851). In a particularlypreferred embodiment, the recombinant cell is a P3X63Ab8.653 or aSP2/0-Ag14 cell.

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to, anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (U.S. Pat. No. 5,266,491), at least one human immunoglobulinpromoter; an enhancer, and/or processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra.Other cells useful for production of nucleic acids or proteins of thepresent invention are known and/or available, for instance, from theAmerican Type Culture Collection Catalogue of Cell Lines and Hybridomas(www.atcc.org) or other known or commercial sources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an Antibody

An anti-IL-12 antibody can be recovered and purified from recombinantcell cultures by well-known methods including, but not limited to,protein A purification, ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. High performance liquid chromatography (“HPLC”) can alsobe employed for purification. See, e.g., Colligan, Current Protocols inImmunology, or Current Protocols in Protein Science, John Wiley & Sons,NY, N.Y., (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirelyincorporated herein by reference.

Antibodies of the present invention include naturally purified products,products of chemical synthetic procedures, and products produced byrecombinant techniques from a eukaryotic host, including, for example,yeast, higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated, withglycosylated preferred. Such methods are described in many standardlaboratory manuals, such as Sambrook, supra, Sections 17.37-17.42;Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, ProteinScience, supra, Chapters 12-14, all entirely incorporated herein byreference.

Anti-IL-12 Antibodies.

An anti-IL-12 antibody according to the present invention includes anyprotein or peptide containing molecule that comprises at least a portionof an immunoglobulin molecule, such as but not limited to, at least oneligand binding portion (LBP), such as but not limited to, acomplementarity determining region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a framework region (e.g., FR1, FR2, FR3, FR4 or fragmentthereof, further optionally comprising at least one substitution,insertion or deletion), a heavy chain or light chain constant region,(e.g., comprising at least one C_(H)1, hinge1, hinge2, hinge3, hinge4,C_(H)2, or C_(H)3 or fragment thereof, further optionally comprising atleast one substitution, insertion or deletion), or any portion thereof,that can be incorporated into an antibody of the present invention. Anantibody of the invention can include or be derived from any mammal,such as but not limited to, a human, a mouse, a rabbit, a rat, a rodent,a primate, or any combination thereof, and the like.

The isolated antibodies of the present invention comprise the antibodyamino acid sequences disclosed herein encoded by any suitablepolynucleotide, or any isolated or prepared antibody. Preferably, thehuman antibody or antigen-binding fragment binds human IL-12 and,thereby, partially or substantially neutralizes at least one biologicalactivity of the protein. An antibody, or specified portion or variantthereof, that partially or preferably substantially neutralizes at leastone biological activity of at least one IL-12 protein or fragment canbind the protein or fragment and thereby inhibit activities mediatedthrough the binding of IL-12 to the IL-12 receptor or through otherIL-12-dependent or mediated mechanisms. As used herein, the term“neutralizing antibody” refers to an antibody that can inhibit anIL-12-dependent activity by about 20-120%, preferably by at least about10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, 100% or more depending on the assay. The capacity of ananti-IL-12 antibody to inhibit an IL-12-dependent activity is preferablyassessed by at least one suitable IL-12 protein or receptor assay, asdescribed herein and/or as known in the art. A human antibody of theinvention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotypeand can comprise a kappa or lambda light chain. In one embodiment, thehuman antibody comprises an IgG heavy chain or defined fragment, forexample, at least one of isotypes, IgG1, IgG2, IgG3 or IgG4 (e.g., γ1,γ2, γ3, γ4). Antibodies of this type can be prepared by employing atransgenic mouse or other trangenic non-human mammal comprising at leastone human light chain (e.g., IgG, IgA, and IgM) transgenes as describedherein and/or as known in the art. In another embodiment, the anti-humanIL-12 human antibody comprises an IgG1 heavy chain and an IgG1 lightchain.

At least one antibody of the invention binds at least one specifiedepitope specific to at least one IL-12 protein, subunit, fragment,portion or any combination thereof. The at least one epitope cancomprise at least one antibody binding region that comprises at leastone portion of the protein, which epitope is preferably comprised of atleast one extracellular, soluble, hydrophillic, external or cytoplasmicportion of the protein. The at least one specified epitope can compriseany combination of at least one amino acid sequence of at least 1-3amino acids to the entire specified portion of contiguous amino acids ofSEQ ID NO:9, for example, amino acid residues 15, 17-21, 23, 40-43,45-47, 54-56 and 58-62.

Generally, the human antibody or antigen-binding fragment of the presentinvention will comprise an antigen-binding region that comprises atleast one human complementarity determining region (CDR1, CDR2 and CDR3)or variant of at least one heavy chain variable region and at least onehuman complementarity determining region (CDR1, CDR2 and CDR3) orvariant of at least one light chain variable region. The CDR sequencesmay be derived from human germline sequences or closely match thegermline sequences. For example, the CDRs from a synthetic libraryderived from the original non-human CDRs can be used. These CDRs may beformed by incorporation of conservative substitutions from the originalnon-human sequence. As a non-limiting example, the antibody orantigen-binding portion or variant can comprise at least one of theheavy chain CDR3 having an amino acid sequence selected from the groupconsisting of SEQ ID NOS:1-3, and/or a light chain CDR3 having an aminoacid sequence selected from the group consisting of SEQ ID NOS:4-6. In aparticular embodiment, the antibody or antigen-binding fragment can havean antigen-binding region that comprises at least a portion of at leastone heavy chain CDR (i.e., CDR1, CDR2 and/or CDR3) having the amino acidsequence of the corresponding CDRs 1, 2, and/or 3 (e.g., SEQ ID NOS:1,2, and/or 3). In another particular embodiment, the antibody orantigen-binding portion or variant can have an antigen-binding regionthat comprises at least a portion of at least one light chain CDR (i.e.,CDR1, CDR2 and/or CDR3) having the amino acid sequence of thecorresponding CDRs 1, 2 and/or 3.

Such antibodies can be prepared by chemically joining together thevarious portions (e.g., CDRs, framework) of the antibody usingconventional techniques, by preparing and expressing a (i.e., one ormore) nucleic acid molecule that encodes the antibody using conventionaltechniques of recombinant DNA technology or by using any other suitablemethod.

The anti-IL-12 antibody can comprise at least one of a heavy or lightchain variable region having a defined amino acid sequence. For example,in a preferred embodiment, the anti-IL-12 antibody comprises at leastone of at least one heavy chain variable region, optionally having theamino acid sequence of SEQ ID NO:7 and/or at least one light chainvariable region, optionally having the amino acid sequence of SEQ IDNO:8. Antibodies that bind to human IL-12 and that comprise a definedheavy or light chain variable region can be prepared using suitablemethods, such as phage display (Katsube, Y., et al., Int J Mol. Med,1(5):863-868 (1998)) or methods that employ transgenic animals, as knownin the art and/or as described herein. For example, a transgenic mouse,comprising a functionally rearranged human immunoglobulin heavy chaintransgene and a transgene comprising DNA from a human immunoglobulinlight chain locus that can undergo functional rearrangement, can beimmunized with human IL-12 or a fragment thereof to elicit theproduction of antibodies. If desired, the antibody producing cells canbe isolated and hybridomas or other immortalized antibody-producingcells can be prepared as described herein and/or as known in the art.Alternatively, the antibody, specified portion or variant can beexpressed using the encoding nucleic acid or portion thereof in asuitable host cell.

The invention also relates to antibodies, antigen-binding fragments,immunoglobulin chains and CDRs comprising amino acids in a sequence thatis substantially the same as an amino acid sequence described herein.Preferably, such antibodies or antigen-binding fragments and antibodiescomprising such chains or CDRs can bind human IL-12 with high affinity(e.g., K_(D) less than or equal to about 10⁻⁹ M) Amino acid sequencesthat are substantially the same as the sequences described hereininclude sequences comprising conservative amino acid substitutions, aswell as amino acid deletions and/or insertions. A conservative aminoacid substitution refers to the replacement of a first amino acid by asecond amino acid that has chemical and/or physical properties (e.g.,charge, structure, polarity, hydrophobicity/hydrophilicity) that aresimilar to those of the first amino acid. Conservative substitutionsinclude, without limitation, replacement of one amino acid by anotherwithin the following groups: lysine (K), arginine (R) and histidine (H);aspartate (D) and glutamate (E); asparagine (N), glutamine (Q), serine(S), threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine(V), leucine (L), isoleucine (I), proline (P), phenylalanine (F),tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W andY; C, S and T.

Amino Acid Codes

The amino acids that make up anti-IL-12 antibodies of the presentinvention are often abbreviated. The amino acid designations can beindicated by designating the amino acid by its single letter code, itsthree letter code, name, or three nucleotide codon(s) as is wellunderstood in the art (see Alberts, B., et al., Molecular Biology of TheCell, Third Ed., Garland Publishing, Inc., New York, 1994):

SINGLE THREE LETTER LETTER THREE NUCLEOTIDE CODE CODE NAME CODON(S) AAla Alanine GCA, GCC, GCG, GCU C Cys Cysteine UGC, UGU D AspAspartic acid GAC, GAU E Glu Glutamic acid GAA, GAG F Phe PhenylanineUUC, UUU G Gly Glycine GGA, GGC, GGG, GGU H His Histidine CAC, CAU I IleIsoleucine AUA, AUC, AUU K Lys Lysine AAA, AAG L Leu LeucineUUA, UUG, CUA, CUC, CUG, CUU M Met Methionine AUG N Asn AsparagineAAC, AAU P Pro Proline CCA, CCC, CCG, CCU Q Gln Glutamine CAA, CAG R ArgArginine AGA, AGG, CGA, CGC, CGG, CGU S Ser Serine AGC, AGU, UCA,UCC, UCG, UCU T Thr Threonine ACA, ACC, ACG, ACU V Val ValineGUA, GUC, GUG, GUU W Trp Tryptophan UGG Y Tyr Tyrosine UAC, UAUAn anti-IL-12 antibody of the present invention can include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation, as specified herein.

The number of amino acid substitutions a skilled artisan would makedepends on many factors, including those described above. Generallyspeaking, the number of amino acid substitutions, insertions ordeletions for any given anti-IL-12 antibody, fragment or variant willnot be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, asspecified herein.

Amino acids in an anti-IL-12 antibody of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g.,Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science244:1081-1085 (1989)). The latter procedure introduces single alaninemutations at every residue in the molecule. The resulting mutantmolecules are then tested for biological activity, such as, but notlimited to, at least one IL-12 neutralizing activity. Sites that arecritical for antibody binding can also be identified by structuralanalysis, such as crystallization, nuclear magnetic resonance orphotoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992)and de Vos, et al., Science 255:306-312 (1992)). The residues on theIL-12p40 antibody involved in IL-12 binding have been identified basedupon the co-crystal structure of the IL-12 antibody and IL-12 p40antigen. These are shown in Table 5 below.

Anti-IL-12 antibodies of the present invention can include, but are notlimited to, at least one portion, sequence or combination selected from5 to all of the contiguous amino acids of at least one of SEQ ID NOS:1,2, 3, 4, 5, 6.

IL-12 antibodies or specified portions or variants of the presentinvention can include, but are not limited to, at least one portion,sequence or combination selected from at least 3-5 contiguous aminoacids of SEQ ID NO:1, 5-17 contiguous amino acids of SEQ ID NO:2, 5-10contiguous amino acids of SEQ ID NO:3, 5-11 contiguous amino acids ofSEQ ID NO:4, 5-7 contiguous amino acids of SEQ ID NO:5; 5-9 contiguousamino acids of SEQ ID NO:6; Leu21, Lys76, Met83, Ser85 of SEQ ID NO:7.

An anti-IL-12 antibody can further optionally comprise a polypeptide ofat least one of 70-100% of 5, 17, 10, 11, 7, 9, 119, or 108 contiguousamino acids of at least one of SEQ ID NOS:1, 2, 3, 4, 5, 6, 7 or 8. Inone embodiment, the amino acid sequence of an immunoglobulin chain, orportion thereof (e.g., variable region, CDR) has about 70-100% identity(e.g., 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85,86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any rangeor value therein) to the amino acid sequence of the corresponding chainof at least one of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7 or 8. For example,the amino acid sequence of a light chain variable region can be comparedwith the sequence of SEQ ID NOS: 4, 5, 6, or 8, or the amino acidsequence of a heavy chain CDR3 can be compared with SEQ ID NOS: 1, 2, 3,or 7. Preferably, 70-100% amino acid identity (i.e., 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or any range or value therein) is determinedusing a suitable computer algorithm, as known in the art.

“Identity,” as known in the art, is a relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, “identity” also meansthe degree of sequence relatedness between polypeptide or polynucleotidesequences, as determined by the match between strings of such sequences.“Identity” and “similarity” can be readily calculated by known methods,including, but not limited to, those described in ComputationalMolecular Biology, Lesk, A. M., ed., Oxford University Press, New York,1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993; Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman,D., Siam J. Applied Math., 48:1073 (1988). In addition, values forpercentage identity can be obtained from amino acid and nucleotidesequence alignments generated using the default settings for the AlignXcomponent of Vector NTI Suite 8.0 (Informax, Frederick, Md.).

Preferred methods to determine identity are designed to give the largestmatch between the sequences tested. Methods to determine identity andsimilarity are codified in publicly available computer programs.Preferred computer program methods to determine identity and similaritybetween two sequences include, but are not limited to, the GCG programpackage (Devereux, J., et al., Nucleic Acids Research 12(1): 387(1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J. Molec.Biol. 215:403-410 (1990)). The BLAST X program is publicly availablefrom NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBINLMNIH Bethesda, Md. 20894: Altschul, S., et al., J. Mol. Biol. 215:403-410(1990). The well-known Smith Waterman algorithm may also be used todetermine identity.

Preferred parameters for polypeptide sequence comparison include thefollowing:

(1) Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970)

Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl.Acad. Sci, USA. 89:10915-10919 (1992)

Gap Penalty: 12

Gap Length Penalty: 4

A program useful with these parameters is publicly available as the“gap” program from Genetics Computer Group, Madison Wis. Theaforementioned parameters are the default parameters for peptidesequence comparisons (along with no penalty for end gaps).

Preferred parameters for polynucleotide comparison include thefollowing:

(1) Algorithm: Needleman and Wunsch, J. Mol Biol. 48:443-453 (1970)

Comparison matrix: matches=+10, mismatch=0

Gap Penalty: 50

Gap Length Penalty: 3

Available as: The “gap” program from Genetics Computer Group, MadisonWis. These are the default parameters for nucleic acid sequencecomparisons.

By way of example, a polynucleotide sequence may be identical to apolynucleotide sequence, that is be 100% identical, or it may include upto a certain integer number of nucleotide alterations as compared to thereference sequence. Such alterations are selected from the groupconsisting of at least one nucleotide deletion, substitution, includingtransition and transversion, or insertion, and wherein the alterationsmay occur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among the nucleotides in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of nucleotide alterations is determined by multiplying the totalnumber of nucleotides in the sequence to be compared by the numericalpercent of the respective percent identity (divided by 100) andsubtracting that product from the total number of nucleotides in SEQ IDNO: A, or:

n.sub.n.ltorsim.x.sub.n-(x.sub.n.y),

wherein n.sub.n is the number of nucleotide alterations, x.sub.n is thetotal number of nucleotides in SEQ ID NO: A, and y is, for instance,0.70 for 70%, 0.80 for 80%, 0.85 for 85%, 0.90 for 90%, 0.95 for 95%,etc., and wherein any non-integer product of x.sub.n and y is roundeddown to the nearest integer prior to subtracting from x.sub.n.

Alterations of a polynucleotide sequence encoding the polypeptide of SEQID NO: 7 may create nonsense, missense or frameshift mutations in thiscoding sequence and thereby alter the polypeptide encoded by thepolynucleotide following such alterations. Similarly, a polypeptidesequence may be identical to the reference sequence of SEQ ID NO: 7,that is be 100% identical, or it may include up to a certain integernumber of amino acid alterations as compared to the reference sequencesuch that the percentage identity is less than 100%. Such alterationsare selected from the group consisting of at least one amino aciddeletion, substitution, including conservative and non-conservativesubstitution, or insertion, and wherein the alterations may occur at theamino- or carboxy-terminal positions of the reference polypeptidesequence or anywhere between those terminal positions, interspersedeither individually among the amino acids in the reference sequence orin one or more contiguous groups within the reference sequence. Thenumber of amino acid alterations for a given % identity is determined bymultiplying the total number of amino acids in SEQ ID NO: 7 by thenumerical percent of the respective percent identity (divided by 100)and then subtracting that product from the total number of amino acidsin SEQ ID NO: 7, or:

n.sub.a.ltorsim.x.sub.a-(x.sub.a.y),

wherein n.sub.a is the number of amino acid alterations, x.sub.a is thetotal number of amino acids in SEQ ID NO: 7, and y is, for instance 0.70for 70%, 0.80 for 80%, 0.85 for 85% etc., and wherein any non-integerproduce of x.sub.a and y is rounded down to the nearest integer prior tosubtracting it from x.sub.a.

Exemplary heavy chain and light chain variable regions sequences andportions thereof are provided in SEQ ID NOS:1-8. The antibodies of thepresent invention, or specified variants thereof, can comprise anynumber of contiguous amino acid residues from an antibody of the presentinvention, wherein that number is selected from the group of integersconsisting of from 10-100% of the number of contiguous residues in ananti-IL-12 antibody. Optionally, this subsequence of contiguous aminoacids is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 ormore amino acids in length, or any range or value therein. Further, thenumber of such subsequences can be any integer selected from the groupconsisting of from 1 to 20, such as at least 2, 3, 4, or 5.

As those of skill will appreciate, the present invention includes atleast one biologically active antibody of the present invention.Biologically active antibodies have a specific activity at least 20%,30%, or 40%, and, preferably, at least 50%, 60%, or 70%, and, mostpreferably, at least 80%, 90%, or 95%-100% or more (including, withoutlimitation, up to 10 times the specific activity) of that of the native(non-synthetic), endogenous or related and known antibody. Methods ofassaying and quantifying measures of enzymatic activity and substratespecificity are well known to those of skill in the art.

Competitive assays can be performed with the antibody of the presentinvention in order to determine what proteins, antibodies, and otherantagonists compete for binding to IL-12p40 with the antibody of thepresent invention and/or share the epitope region. These assays asreadily known to those of ordinary skill in the art evaluate competitionbetween antagonists or ligands for a limited number of binding sites ona protein, e.g., p40. The protein and/or antibody is immobilized orinsolubilized before or after the competition and the sample bound tothe p40 subunit is separated from the unbound sample, for example, bydecanting (where the protein/antibody was preinsolubilized) or bycentrifuging (where the protein/antibody was precipitated after thecompetitive reaction). Also, the competitive binding may be determinedby whether function is altered by the binding or lack of binding of theantibody to the protein, e.g., whether the antibody molecule inhibits orpotentiates the enzymatic activity of, for example, a label. ELISA andother functional assays may be used, as well known in the art.

In another aspect, the invention relates to human antibodies andantigen-binding fragments, as described herein, which are modified bythe covalent attachment of an organic moiety. Such modification canproduce an antibody or antigen-binding fragment with improvedpharmacokinetic properties (e.g., increased in vivo serum half-life).The organic moiety can be a linear or branched hydrophilic polymericgroup, fatty acid group, or fatty acid ester group. In particularembodiments, the hydrophilic polymeric group can have a molecular weightof about 800 to about 120,000 Daltons and can be a polyalkane glycol(e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)),carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, andthe fatty acid or fatty acid ester group can comprise from about eightto about forty carbon atoms.

The modified antibodies and antigen-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the antibody. Each organic moiety that isbonded to an antibody or antigen-binding fragment of the invention canindependently be a hydrophilic polymeric group, a fatty acid group or afatty acid ester group. As used herein, the term “fatty acid”encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anantibody modified by the covalent attachment of polylysine isencompassed by the invention. Hydrophilic polymers suitable formodifying antibodies of the invention can be linear or branched andinclude, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, polysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies theantibody of the invention has a molecular weight of about 800 to about150,000 Daltons as a separate molecular entity. For example, PEG₅₀₀₀ andPEG_(20,000), wherein the subscript is the average molecular weight ofthe polymer in Daltons, can be used. The hydrophilic polymeric group canbe substituted with one to about six alkyl, fatty acid or fatty acidester groups. Hydrophilic polymers that are substituted with a fattyacid or fatty acid ester group can be prepared by employing suitablemethods. For example, a polymer comprising an amine group can be coupledto a carboxylate of the fatty acid or fatty acid ester, and an activatedcarboxylate (e.g., activated with N,N-carbonyl diimidazole) on a fattyacid or fatty acid ester can be coupled to a hydroxyl group on apolymer.

Fatty acids and fatty acid esters suitable for modifying antibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying antibodies ofthe invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,11,14-eicosatetraenoate (C₂₀, arachidonate),octanedioic acid, tetradecanedioic acid, octadecanedioic acid,docosanedioic acid, and the like. Suitable fatty acid esters includemono-esters of dicarboxylic acids that comprise a linear or branchedlower alkyl group. The lower alkyl group can comprise from one to abouttwelve, preferably, one to about six, carbon atoms.

The modified human antibodies and antigen-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups, such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example, adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom, such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate, as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221, the entire teachings of which areincorporated herein by reference.)

The modified antibodies of the invention can be produced by reacting ahuman antibody or antigen-binding fragment with a modifying agent. Forexample, the organic moieties can be bonded to the antibody in anon-site specific manner by employing an amine-reactive modifying agent,for example, an NHS ester of PEG. Modified human antibodies orantigen-binding fragments can also be prepared by reducing disulfidebonds (e.g., intra-chain disulfide bonds) of an antibody orantigen-binding fragment. The reduced antibody or antigen-bindingfragment can then be reacted with a thiol-reactive modifying agent toproduce the modified antibody of the invention. Modified humanantibodies and antigen-binding fragments comprising an organic moietythat is bonded to specific sites of an antibody of the present inventioncan be prepared using suitable methods, such as reverse proteolysis(Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996);Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and themethods described in Hermanson, G. T., Bioconjugate Techniques, AcademicPress: San Diego, Calif. (1996).

Anti-Idiotype Antibodies to Anti-IL-12 Antibody Compositions

In addition to monoclonal anti-IL-12 antibodies, the present inventionis also directed to an anti-idiotypic (anti-Id) antibody specific forsuch antibodies of the invention. An anti-Id antibody is an antibodywhich recognizes unique determinants generally associated with theantigen-binding region of another antibody. The anti-Id can be preparedby immunizing an animal of the same species and genetic type (e.g.,mouse strain) as the source of the Id antibody with the antibody or aCDR containing region thereof. The immunized animal will recognize andrespond to the idiotypic determinants of the immunizing antibody andproduce an anti-Id antibody. The anti-Id antibody may also be used as an“immunogen” to induce an immune response in yet another animal,producing a so-called anti-anti-Id antibody.

The present invention also provides at least one anti-IL-12 antibodycomposition comprising at least one, at least two, at least three, atleast four, at least five, at least six or more anti-IL-12 antibodiesthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcompositions comprise non-naturally occurring compositions comprising atleast one or two full length, C- and/or N-terminally deleted variants,domains, fragments, or specified variants, of the anti-IL-12 antibodyamino acid sequence selected from the group consisting of 70-100% of thecontiguous amino acids of SEQ ID NOS: 7 or 8, or specified fragments,domains or variants thereof. Preferred anti-IL-12 antibody compositionsinclude at least one or two full length, fragments, domains or variantsas at least one CDR or LBP containing portions of the anti-IL-12antibody sequence described herein, for example, 70-100% of SEQ IDNOS:1-6, 7, or 8, or specified fragments, domains or variants thereof.Further preferred compositions comprise, for example, 40-99% of at leastone of 70-100% of SEQ ID NOS: 1-6, 7, or 8, etc., or specifiedfragments, domains or variants thereof. Such composition percentages areby weight, volume, concentration, molarity, or molality as liquid or drysolutions, mixtures, suspension, emulsions, particles, powder, orcolloids, as known in the art or as described herein.

Antibody Compositions Comprising Further Therapeutically ActiveIngredients

The antibody compositions of the invention can optionally furthercomprise an effective amount of at least one compound or proteinselected from at least one of an anti-infective drug, a cardiovascular(CV) system drug, a central nervous system (CNS) drug, an autonomicnervous system (ANS) drug, a respiratory tract drug, a gastrointestinal(GI) tract drug, a hormonal drug, a drug for fluid or electrolytebalance, a hematologic drug, an antineoplastic, an immunomodulationdrug, an ophthalmic, otic or nasal drug, a topical drug, a nutritionaldrug or the like. Such drugs are well known in the art, includingformulations, indications, dosing and administration for each presentedherein (see, e.g., Nursing 2001 Handbook of Drugs, 21^(st) edition,Springhouse Corp., Springhouse, Pa., 2001; Health Professional's DrugGuide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, UpperSaddle River, N.J.; Pharmcotherapy Handbook, Wells et al., ed., Appleton& Lange, Stamford, Conn., each entirely incorporated herein byreference).

The anti-infective drug can be at least one selected from amebicides orat least one antiprotozoals, anthelmintics, antifungals, antimalarials,antituberculotics or at least one antileprotics, aminoglycosides,penicillins, cephalosporins, tetracyclines, sulfonamides,fluoroquinolones, antivirals, macrolide anti-infectives, andmiscellaneous anti-infectives. The CV drug can be at least one selectedfrom inotropics, antiarrhythmics, antianginals, antihypertensives,antilipemics, and miscellaneous cardiovascular drugs. The CNS drug canbe at least one selected from normarcotic analgesics or at least oneselected from antipyretics, nonsteroidal anti-inflammatory drugs,narcotic or at least one opioid analgesics, sedative-hypnotics,anticonvulsants, antidepressants, antianxiety drugs, antipsychotics,central nervous system stimulants, antiparkinsonians, and miscellaneouscentral nervous system drugs. The ANS drug can be at least one selectedfrom cholinergics (parasympathomimetics), anticholinergics, adrenergics(sympathomimetics), adrenergic blockers (sympatholytics), skeletalmuscle relaxants, and neuromuscular blockers. The respiratory tract drugcan be at least one selected from antihistamines, bronchodilators,expectorants or at least one antitussive, and miscellaneous respiratorydrugs. The GI tract drug can be at least one selected from antacids orat least one adsorbent or at least one antiflatulent, digestive enzymeor at least one gallstone solubilizer, antidiarrheals, laxatives,antiemetics, and antiulcer drugs. The hormonal drug can be at least oneselected from corticosteroids, androgens or at least one anabolicsteroid, estrogen or at least one progestin, gonadotropin, antidiabeticdrug or at least one glucagon, thyroid hormone, thyroid hormoneantagonist, pituitary hormone, and parathyroid-like drug. The drug forfluid and electrolyte balance can be at least one selected fromdiuretics, electrolytes or at least one replacement solution, acidifieror at least one alkalinizer. The hematologic drug can be at least oneselected from hematinics, anticoagulants, blood derivatives, andthrombolytic enzymes. The antineoplastics can be at least one selectedfrom alkylating drugs, antimetabolites, antibiotic antineoplastics,antineoplastics that alter hormone balance, and miscellaneousantineoplastics. The immunomodulation drug can be at least one selectedfrom immunosuppressants, vaccines or at least one toxoid, antitoxin orat least one antivenin, immune serum, and biological response modifier.The ophthalmic, otic, and nasal drugs can be at least one selected fromophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics,mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,otics, and nasal drugs. The topical drug can be at least one selectedfrom local anti-infectives, scabicides or at least one pediculicide ortopical corticosteroid. The nutritional drug can be at least oneselected from vitamins, minerals, or calorics. See, e.g., contents ofNursing 2001 Drug Handbook, supra.

The at least one amebicide or antiprotozoal can be at least one selectedfrom atovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, and pentamidine isethionate.The at least one anthelmintic can be at least one selected frommebendazole, pyrantel pamoate, and thiabendazole. The at least oneantifungal can be at least one selected from amphotericin B,amphotericin B cholesteryl sulfate complex, amphotericin B lipidcomplex, amphotericin B liposomal, fluconazole, flucytosine,griseofulvin microsize, griseofulvin ultramicrosize, itraconazole,ketoconazole, nystatin, and terbinafine hydrochloride. The at least oneantimalarial can be at least one selected from chloroquinehydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquinesulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine,and pyrimethamine with sulfadoxine. The at least one antituberculotic orantileprotic can be at least one selected from clofazimine, cycloserine,dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin,rifampin, rifapentine, and streptomycin sulfate. The at least oneaminoglycoside can be at least one selected from amikacin sulfate,gentamicin sulfate, neomycin sulfate, streptomycin sulfate, andtobramycin sulfate. The at least one penicillin can be at least oneselected from amoxcillin/clavulanate potassium, amoxicillin trihydrate,ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillinsodium/sulbactam sodium, cloxacillin sodium, dicloxacillin sodium,mezlocillin sodium, nafcillin sodium, oxacillin sodium, penicillin Gbenzathine, penicillin G potassium, penicillin G procaine, penicillin Gsodium, penicillin V potassium, piperacillin sodium, piperacillinsodium/tazobactam sodium, ticarcillin disodium, and ticarcillindisodium/clavulanate potassium. The at least one cephalosporin can be atleast one selected from cefaclor, cefadroxil, cefazolin sodium,cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium,cefonicid sodium, cefoperazone sodium, cefotaxime sodium, cefotetandisodium, cefoxitin sodium, cefpodoxime proxetil, cefprozil,ceftazidime, ceftibuten, ceftizoxime sodium, ceftriaxone sodium,cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride,cephalexin monohydrate, cephradine, and loracarbef. The at least onetetracycline can be at least one selected from demeclocyclinehydrochloride, doxycycline calcium, doxycycline hyclate, doxycyclinehydrochloride, doxycycline monohydrate, minocycline hydrochloride, andtetracycline hydrochloride. The at least one sulfonamide can be at leastone selected from co-trimoxazole, sulfadiazine, sulfamethoxazole,sulfisoxazole, and sulfisoxazole acetyl. The at least onefluoroquinolone can be at least one selected from alatrofloxacinmesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacinhydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, andtrovafloxacin mesylate. The at least one fluoroquinolone can be at leastone selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin,levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,ofloxacin, sparfloxacin, and trovafloxacin mesylate. The at least oneantiviral can be at least one selected from abacavir sulfate, acyclovirsodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdinemesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium,foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine,lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivirphosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir,saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine,zanamivir, and zidovudine. The at least one macroline anti-infective canbe at least one selected from azithromycin, clarithromycin,dirithromycin, erythromycin base, erythromycin estolate, erythromycinethylsuccinate, erythromycin lactobionate, and erythromycin stearate.The at least one miscellaneous anti-infective can be at least oneselected from aztreonam, bacitracin, chloramphenicol sodium sucinate,clindamycin hydrochloride, clindamycin palmitate hydrochloride,clindamycin phosphate, imipenem and cilastatin sodium, meropenem,nitrofurantoin macrocrystals, nitrofurantoin microcrystals,quinupristin/dalfopristin, spectinomycin hydrochloride, trimethoprim,and vancomycin hydrochloride. (See, e.g., pp. 24-214 of Nursing 2001Drug Handbook.)

The at least one inotropic can be at least one selected from aminonelactate, digoxin, and milrinone lactate. The at least one antiarrhythmiccan be at least one selected from adenosine, amiodarone hydrochloride,atropine sulfate, bretylium tosylate, diltiazem hydrochloride,disopyramide, disopyramide phosphate, esmolol hydrochloride, flecamideacetate, ibutilide fumarate, lidocaine hydrochloride, mexiletinehydrochloride, moricizine hydrochloride, phenyloin, phenyloin sodium,procainamide hydrochloride, propafenone hydrochloride, propranololhydrochloride, quinidine bisulfate, quinidine gluconate, quinidinepolygalacturonate, quinidine sulfate, sotalol, tocamide hydrochloride,and verapamil hydrochloride. The at least one antianginal can be atleast one selected from amlodipidine besylate, amyl nitrite, bepridilhydrochloride, diltiazem hydrochloride, isosorbide dinitrate, isosorbidemononitrate, nadolol, nicardipine hydrochloride, nifedipine,nitroglycerin, propranolol hydrochloride, verapamil, and verapamilhydrochloride. The at least one antihypertensive can be at least oneselected from acebutolol hydrochloride, amlodipine besylate, atenolol,benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol,clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride,doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate,felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate,guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride,irbesartan, isradipine, labetalol hydrchloride, lisinopril, losartanpotassium, methyldopa, methyldopate hydrochloride, metoprolol succinate,metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol,nicardipine hydrochloride, nifedipine, nisoldipine, nitroprussidesodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate,pindolol, prazosin hydrochloride, propranolol hydrochloride, quinaprilhydrochloride, ramipril, telmisartan, terazosin hydrochloride, timololmaleate, trandolapril, valsartan, and verapamil hydrochloride. The atleast one antilipemic can be at least one selected from atorvastatincalcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride,fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin,niacin, pravastatin sodium, and simvastatin. The at least onemiscellaneous CV drug can be at least one selected from abciximab,alprostadil, arbutamine hydrochloride, cilostazol, clopidogrelbisulfate, dipyridamole, eptifibatide, midodrine hydrochloride,pentoxifylline, ticlopidine hydrochloride, and tirofiban hydrochloride.(See, e.g., pp. 215-336 of Nursing 2001 Drug Handbook.)

The at least one normarcotic analgesic or antipyretic can be at leastone selected from acetaminophen, aspirin, choline magnesiumtrisalicylate, diflunisal, and magnesium salicylate. The at least onenonsteroidal anti-inflammatory drug can be at least one selected fromcelecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofencalcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodiumtrihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen,naproxen sodium, oxaprozin, piroxicam, rofecoxib, and sulindac. The atleast one narcotic or opioid analgesic can be at least one selected fromalfentanil hydrochloride, buprenorphine hydrochloride, butorphanoltartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyltransdermal system, fentanyl transmucosal, hydromorphone hydrochloride,meperidine hydrochloride, methadone hydrochloride, morphinehydrochloride, morphine sulfate, morphine tartrate, nalbuphinehydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymorphonehydrochloride, pentazocine hydrochloride, pentazocine hydrochloride andnaloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride,propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate,and tramadol hydrochloride. The at least one sedative-hypnotic can be atleast one selected from chloral hydrate, estazolam, flurazepamhydrochloride, pentobarbital, pentobarbital sodium, phenobarbitalsodium, secobarbital sodium, temazepam, triazolam, zaleplon, andzolpidem tartrate. The at least one anticonvulsant can be at least oneselected from acetazolamide sodium, carbamazepine, clonazepam,clorazepate dipotassium, diazepam, divalproex sodium, ethosuximde,fosphenyloin sodium, gabapentin, lamotrigine, magnesium sulfate,phenobarbital, phenobarbital sodium, phenyloin, phenyloin sodium,phenyloin sodium (extended), primidone, tiagabine hydrochloride,topiramate, valproate sodium, and valproic acid. The at least oneantidepressant can be at least one selected from amitriptylinehydrochloride, amitriptyline pamoate, amoxapine, bupropionhydrochloride, citalopram hydrobromide, clomipramine hydrochloride,desipramine hydrochloride, doxepin hydrochloride, fluoxetinehydrochloride, imipramine hydrochloride, imipramine pamoate,mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride,paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride,tranylcypromine sulfate, trimipramine maleate, and venlafaxinehydrochloride. The at least one antianxiety drug can be at least oneselected from alprazolam, buspirone hydrochloride, chlordiazepoxide,chlordiazepoxide hydrochloride, clorazepate dipotassium, diazepam,doxepin hydrochloride, hydroxyzine embonate, hydroxyzine hydrochloride,hydroxyzine pamoate, lorazepam, mephrobamate, midazolam hydrochloride,and oxazepam. The at least one antipsychotic drug can be at least oneselected from chlorpromazine hydrochloride, clozapine, fluphenazinedecanoate, fluephenazine enanthate, fluphenazine hydrochloride,haloperidol, haloperidol decanoate, haloperidol lactate, loxapinehydrochloride, loxapine succinate, mesoridazine besylate, molindonehydrochloride, olanzapine, perphenazine, pimozide, prochlorperazine,quetiapine fumarate, risperidone, thioridazine hydrochloride,thiothixene, thiothixene hydrochloride, and trifluoperazinehydrochloride. The at least one central nervous system stimulant can beat least one selected from amphetamine sulfate, caffeine,dextroamphetamine sulfate, doxapram hydrochloride, methamphetaminehydrochloride, methylphenidate hydrochloride, modafinil, pemoline, andphentermine hydrochloride. The at least one antiparkinsonian can be atleast one selected from amantadine hydrochloride, benztropine mesylate,biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,pramipexole dihydrochloride, ropinirole hydrochloride, selegilinehydrochloride, tolcapone, and trihexyphenidyl hydrochloride. The atleast one miscellaneous central nervous system drug can be at least oneselected from bupropion hydrochloride, donepezil hydrochloride,droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate,naratriptan hydrochloride, nicotine polacrilex, nicotine transdermalsystem, propofol, rizatriptan benzoate, sibutramine hydrochloridemonohydrate, sumatriptan succinate, tacrine hydrochloride, andzolmitriptan. (See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.)

The at least one cholinergic (e.g., parasymathomimetic) can be at leastone selected from bethanechol chloride, edrophonium chloride,neostigmine bromide, neostigmine methylsulfate, physostigminesalicylate, and pyridostigmine bromide. The at least one anticholinergiccan be at least one selected from atropine sulfate, dicyclominehydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate,propantheline bromide, scopolamine, scopolamine butylbromide, andscopolamine hydrobromide. The at least one adrenergic (sympathomimetics)can be at least one selected from dobutamine hydrochloride, dopaminehydrochloride, metaraminol bitartrate, norepinephrine bitartrate,phenylephrine hydrochloride, pseudoephedrine hydrochloride, andpseudoephedrine sulfate. The at least one adrenergic blocker(sympatholytic) can be at least one selected from dihydroergotaminemesylate, ergotamine tartrate, methysergide maleate, and propranololhydrochloride. The at least one skeletal muscle relaxant can be at leastone selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprinehydrochloride, dantrolene sodium, methocarbamol, and tizanidinehydrochloride. The at least one neuromuscular blocker can be at leastone selected from atracurium besylate, cisatracurium besylate,doxacurium chloride, mivacurium chloride, pancuronium bromide,pipecuronium bromide, rapacuronium bromide, rocuronium bromide,succinylcholine chloride, tubocurarine chloride, and vecuronium bromide.(See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)

The at least one antihistamine can be at least one selected frombrompheniramine maleate, cetirizine hydrochloride, chlorpheniraminemaleate, clemastine fumarate, cyproheptadine hydrochloride,diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine,promethazine hydrochloride, promethazine theoclate, and triprolidinehydrochloride. The at least one bronchodilator can be at least oneselected from albuterol, albuterol sulfate, aminophylline, atropinesulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate,epinephrine hydrochloride, ipratropium bromide, isoproterenol,isoproterenol hydrochloride, isoproterenol sulfate, levalbuterolhydrochloride, metaproterenol sulfate, oxtriphylline, pirbuterolacetate, salmeterol xinafoate, terbutaline sulfate, and theophylline.The at least one expectorant or antitussive can be at least one selectedfrom benzonatate, codeine phosphate, codeine sulfate, dextramethorphanhydrobromide, diphenhydramine hydrochloride, guaifenesin, andhydromorphone hydrochloride. The at least one miscellaneous respiratorydrug can be at least one selected from acetylcysteine, beclomethasonedipropionate, beractant, budesonide, calfactant, cromolyn sodium,dornase alfa, epoprostenol sodium, flunisolide, fluticasone propionate,montelukast sodium, nedocromil sodium, palivizumab, triamcinoloneacetonide, zafirlukast, and zileuton. (See, e.g., pp. 585-642 of Nursing2001 Drug Handbook.)

The at least one antacid, adsorbent, or antiflatulent can be at leastone selected from aluminum carbonate, aluminum hydroxide, calciumcarbonate, magaldrate, magnesium hydroxide, magnesium oxide,simethicone, and sodium bicarbonate. The at least one digestive enzymeor gallstone solubilizer can be at least one selected from pancreatin,pancrelipase, and ursodiol. The at least one antidiarrheal can be atleast one selected from attapulgite, bismuth subsalicylate, calciumpolycarbophil, diphenoxylate hydrochloride and atropine sulfate,loperamide, octreotide acetate, opium tincture, and opium tincure(camphorated). The at least one laxative can be at least one selectedfrom bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagradaaromatic fluidextract, cascara sagrada fluidextract, castor oil,docusate calcium, docusate sodium, glycerin, lactulose, magnesiumcitrate, magnesium hydroxide, magnesium sulfate, methylcellulose,mineral oil, polyethylene glycol or electrolyte solution, psyllium,senna, and sodium phosphates. The at least one antiemetic can be atleast one selected from chlorpromazine hydrochloride, dimenhydrinate,dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizinehydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride,perphenazine, prochlorperazine, prochlorperazine edisylate,prochlorperazine maleate, promethazine hydrochloride, scopolamine,thiethylperazine maleate, and trimethobenzamide hydrochloride. The atleast one antiulcer drug can be at least one selected from cimetidine,cimetidine hydrochloride, famotidine, lansoprazole, misoprostol,nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate,ranitidine hydrochloride, and sucralfate. (See, e.g., pp. 643-95 ofNursing 2001 Drug Handbook.)

The at least one coricosteroid can be at least one selected frombetamethasone, betamethasone acetate or betamethasone sodium phosphate,betamethasone sodium phosphate, cortisone acetate, dexamethasone,dexamethasone acetate, dexamethasone sodium phosphate, fludrocortisoneacetate, hydrocortisone, hydrocortisone acetate, hydrocortisonecypionate, hydrocortisone sodium phosphate, hydrocortisone sodiumsuccinate, methylprednisolone, methylprednisolone acetate,methylprednisolone sodium succinate, prednisolone, prednisolone acetate,prednisolone sodium phosphate, prednisolone tebutate, prednisone,triamcinolone, triamcinolone acetonide, and triamcinolone diacetate. Theat least one androgen or anabolic steroid can be at least one selectedfrom danazol, fluoxymesterone, methyltestosterone, nandrolone decanoate,nandrolone phenpropionate, testosterone, testosterone cypionate,testosterone enanthate, testosterone propionate, and testosteronetransdermal system. The at least one estrogen or progestin can be atleast one selected from esterified estrogens, estradiol, estradiolcypionate, estradiol/norethindrone acetate transdermal system, estradiolvalerate, estrogens (conjugated), estropipate, ethinyl estradiol,ethinyl estradiol and desogestrel, ethinyl estradiol and ethynodioldiacetate, ethinyl estradiol and desogestrel, ethinyl estradiol andethynodiol diacetate, ethinyl estradiol and levonorgestrel, ethinylestradiol and norethindrone, ethinyl estradiol and norethindroneacetate, ethinyl estradiol and norgestimate, ethinyl estradiol andnorgestrel, ethinyl estradiol and norethindrone and acetate and ferrousfumarate, levonorgestrel, medroxyprogesterone acetate, mestranol andnorethindron, norethindrone, norethindrone acetate, norgestrel, andprogesterone. The at least one gonadroptropin can be at least oneselected from ganirelix acetate, gonadoreline acetate, histrelinacetate, and menotropins. The at least one antidiabetic or glucaon canbe at least one selected from acarbose, chlorpropamide, glimepiride,glipizide, glucagon, glyburide, insulins, metformin hydrochloride,miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazonemaleate, and troglitazone. The at least one thyroid hormone can be atleast one selected from levothyroxine sodium, liothyronine sodium,liotrix, and thyroid. The at least one thyroid hormone antagonist can beat least one selected from methimazole, potassium iodide, potassiumiodide (saturated solution), propylthiouracil, radioactive iodine(sodium iodide ¹³¹I) and strong iodine solution. The at least onepituitary hormone can be at least one selected from corticotropin,cosyntropin, desmophressin acetate, leuprolide acetate, repositorycorticotropin, somatrem, somatropin, and vasopressin. The at least oneparathyroid-like drug can be at least one selected from calcifediol,calcitonin (human), calcitonin (salmon), calcitriol, dihydrotachysterol,and etidronate disodium. (See, e.g., pp. 696-796 of Nursing 2001 DrugHandbook.)

The at least one diuretic can be at least one selected fromacetazolamide, acetazolamide sodium, amiloride hydrochloride,bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid,furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,spironolactone, torsemide, triamterene, and urea. The at least oneelectrolyte or replacement solution can be at least one selected fromcalcium acetate, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, calciumlactate, calcium phosphate (dibasic), calcium phosphate (tribasic),dextran (high-molecular-weight), dextran (low-molecular-weight),hetastarch, magnesium chloride, magnesium sulfate, potassium acetate,potassium bicarbonate, potassium chloride, potassium gluconate, Ringer'sinjection, Ringer's injection (lactated), and sodium chloride. The atleast one acidifier or alkalinizer can be at least one selected fromsodium bicarbonate, sodium lactate, and tromethamine (See, e.g., pp.797-833 of Nursing 2001 Drug Handbook.)

The at least one hematinic can be at least one selected from ferrousfumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried),iron dextran, iron sorbitol, polysaccharide-iron complex, and sodiumferric gluconate complex. The at least one anticoagulant can be at leastone selected from ardeparin sodium, dalteparin sodium, danaparoidsodium, enoxaparin sodium, heparin calcium, heparin sodium, and warfarinsodium. The at least one blood derivative can be at least one selectedfrom albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitorcoagulant complex, antithrombin III (human), factor IX (human), factorIX complex, and plasma protein fractions. The at least one thrombolyticenzyme can be at least one selected from alteplase, anistreplase,reteplase (recombinant), streptokinase, and urokinase. (See, e.g., pp.834-66 of Nursing 2001 Drug Handbook.)

The at least one alkylating drug can be at least one selected frombusulfan, carboplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride,melphalan, melphalan hydrochloride, streptozocin, temozolomide, andthiotepa. The at least one antimetabolite can be at least one selectedfrom capecitabine, cladribine, cytarabine, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate,methotrexate sodium, and thioguanine. The at least one antibioticantineoplastic can be at least one selected from bleomycin sulfate,dactinomycin, daunorubicin citrate liposomal, daunorubicinhydrochloride, doxorubicin hydrochloride, doxorubicin hydrochlorideliposomal, epirubicin hydrochloride, idarubicin hydrochloride,mitomycin, pentostatin, plicamycin, and valrubicin. The at least oneantineoplastic that alters hormone balance can be at least one selectedfrom anastrozole, bicalutamide, estramustine phosphate sodium,exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate,megestrol acetate, nilutamide, tamoxifen citrate, testolactone, andtoremifene citrate. The at least one miscellaneous antineoplastic can beat least one selected from asparaginase, bacillus Calmette-Guerin (BCG)(live intravesical), dacarbazine, docetaxel, etoposide, etoposidephosphate, gemcitabine hydrochloride, irinotecan hydrochloride,mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimersodium, procarbazine hydrochloride, rituximab, teniposide, topotecanhydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristinesulfate, and vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing2001 Drug Handbook.)

The at least one immunosuppressant can be at least one selected fromazathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immuneglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetilhydrochloride, sirolimus, and tacrolimus. The at least one vaccine ortoxoid can be at least one selected from BCG vaccine, cholera vaccine,diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanustoxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanustoxoids and whole-cell pertussis vaccine, Haemophilus b conjugatevaccines, hepatitis A vaccine (inactivated), hepatisis B vaccine(recombinant), influenza virus vaccine 1999-2000 trivalent types A & B(purified surface antigen), influenza virus vaccine 1999-2000 trivalenttypes A & B (subvirion or purified subvirion), influenza virus vaccine1999-2000 trivalent types A & B (whole virion), Japanese encephalitisvirus vaccine (inactivated), Lyme disease vaccine (recombinant OspA),measles and mumps and rubella virus vaccine (live), measles and mumpsand rubella virus vaccine (live attenuated), measles virus vaccine (liveattenuated), meningococcal polysaccharide vaccine, mumps virus vaccine(live), plague vaccine, pneumococcal vaccine (polyvalent), poliovirusvaccine (inactivated), poliovirus vaccine (live, oral, trivalent),rabies vaccine (adsorbed), rabies vaccine (human diploid cell), rubellaand mumps virus vaccine (live), rubella virus vaccine (live,attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoidvaccine (oral), typhoid vaccine (parenteral), typhoid Vi polysaccharidevaccine, varicella virus vaccine, and yellow fever vaccine. The at leastone antitoxin or antivenin can be at least one selected from black widowspider antivenin, Crotalidae antivenom (polyvalent), diphtheriaantitoxin (equine), and Micrurus fulvius antivenin. The at least oneimmune serum can be at least one selected from cytomegalovirus immuneglobulin (intraveneous), hepatitis B immune globulin (human), immuneglobulin intramuscular, immune globulin intravenous, rabies immuneglobulin (human), respiratory syncytial virus immune globulinintravenous (human), Rh₀(D) immune globulin (human), Rh₀(D) immuneglobulin intravenous (human), tetanus immune globulin (human), andvaricella-zoster immune globulin. The at least one biological responsemodifier can be at least one selected from aldesleukin, epoetin alfa,filgrastim, glatiramer acetate for injection, interferon alfacon-1,interferon alfa-2a (recombinant), interferon alfa-2b (recombinant),interferon beta-1a, interferon beta-1b (recombinant), interferongamma-1b, levamisole hydrochloride, oprelvekin, and sargramostim. (See,e.g., pp. 964-1040 of Nursing 2001 Drug Handbook.)

The at least one ophthalmic anti-infective can be selected formbacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin,gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamidesodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%,tobramycin, and vidarabine. The at least one ophthalmicanti-inflammatory can be at least one selected from dexamethasone,dexamethasone sodium phosphate, diclofenac sodium 0.1%, fluorometholone,flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate(suspension) and prednisolone sodium phosphate (solution). The at leastone miotic can be at least one selected from acetylocholine chloride,carbachol (intraocular), carbachol (topical), echothiophate iodide,pilocarpine, pilocarpine hydrochloride, and pilocarpine nitrate. The atleast one mydriatic can be at least one selected from atropine sulfate,cyclopentolate hydrochloride, epinephrine hydrochloride, epinephrylborate, homatropine hydrobromide, phenylephrine hydrochloride,scopolamine hydrobromide, and tropicamide. The at least one ophthalmicvasoconstrictor can be at least one selected from naphazolinehydrochloride, oxymetazoline hydrochloride, and tetrahydrozolinehydrochloride. The at least one miscellaneous ophthalmic can be at leastone selected from apraclonidine hydrochloride, betaxolol hydrochloride,brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride,dorzolamide hydrochloride, emedastine difumarate, fluorescein sodium,ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranololhydrochloride, sodium chloride (hypertonic), and timolol maleate. The atleast one otic can be at least one selected from boric acid, carbamideperoxide, chloramphenicol, and triethanolamine polypeptideoleate-condensate. The at least one nasal drug can be at least oneselected from beclomethasone dipropionate, budesonide, ephedrinesulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate,naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrinehydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide,and xylometazoline hydrochloride. (See, e.g., pp. 1041-97 of Nursing2001 Drug Handbook.)

The at least one local anti-infective can be at least one selected fromacyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazolenitrate, clindamycin phosphate, clotrimazole, econazole nitrate,erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,metronidazole (topical), miconazole nitrate, mupirocin, naftifinehydrochloride, neomycin sulfate, nitrofurazone, nystatin, silversulfadiazine, terbinafine hydrochloride, terconazole, tetracyclinehydrochloride, tioconazole, and tolnaftate. The at least one scabicideor pediculicide can be at least one selected from crotamiton, lindane,permethrin, and pyrethrins. The at least one topical corticosteroid canbe at least one selected from betamethasone dipropionate, betamethasonevalerate, clobetasol propionate, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasonepropionate, halcionide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate, andtriamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 DrugHandbook.)

The at least one vitamin or mineral can be at least one selected fromvitamin A, vitamin B complex, cyanocobalamin, folic acid,hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxinehydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D,cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol,paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodiumfluoride, sodium fluoride (topical), trace elements, chromium, copper,iodine, manganese, selenium, and zinc. The at least one caloric can beat least one selected from amino acid infusions (crystalline), aminoacid infusions in dextrose, amino acid infusions with electrolytes,amino acid infusions with electrolytes in dextrose, amino acid infusionsfor hepatic failure, amino acid infusions for high metabolic stress,amino acid infusions for renal failure, dextrose, fat emulsions, andmedium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 DrugHandbook.)

Anti-IL-12 antibody compositions of the present invention can furthercomprise at least one of any suitable and effective amount of acomposition or pharmaceutical composition comprising at least oneanti-IL-12 antibody contacted or administered to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy,optionally further comprising at least one selected from at least oneTNF antagonist (e.g., but not limited to a TNF chemical or proteinantagonist, TNF monoclonal or polyclonal antibody or fragment, a solubleTNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptidesthereof, or a small molecule TNF antagonist, e.g., TNF binding protein Ior II (TBP-1 or TBP-II), nerelimonmab, infliximab, eternacept, CDP-571,CDP-870, afelimomab, lenercept, and the like), an antirheumatic (e.g.,methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, goldsodium thiomalate, hydroxychloroquine sulfate, leflunomide,sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluororquinolone, a macrolide, apenicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropoietin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Non-limiting examplesof such cytokines include, but are not limited to, any of IL-1 to IL-23(e.g., IL-1, IL-2, etc.). Suitable dosages are well known in the art.See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

Such anti-cancer or anti-infectives can also include toxin moleculesthat are associated, bound, co-formulated or co-administered with atleast one antibody of the present invention. The toxin can optionallyact to selectively kill the pathologic cell or tissue. The pathologiccell can be a cancer or other cell. Such toxins can be, but are notlimited to, purified or recombinant toxin or toxin fragment comprisingat least one functional cytotoxic domain of toxin, e.g., selected fromat least one of ricin, diphtheria toxin, a venom toxin, or a bacterialtoxin. The term toxin also includes both endotoxins and exotoxinsproduced by any naturally occurring, mutant or recombinant bacteria orviruses which may cause any pathological condition in humans and othermammals, including toxin shock, which can result in death. Such toxinsmay include, but are not limited to, enterotoxigenic E. coli heat-labileenterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin,Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-1),Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcalenterotoxins and the like. Such bacteria include, but are not limitedto, strains of a species of enterotoxigenic E. coli (ETEC),enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcuspyogenes), Shigella species (e.g., Shigella dysenteriae, Shigellaflexneri, Shigella boydii, and Shigella sonnei), Salmonella species(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonellaenteritidis), Clostridium species (e.g., Clostridium perfringens,Clostridium dificile, Clostridium botulinum), Camphlobacter species(e.g., Camphlobacter jejuni, Camphlobacter fetus), Heliobacter species,(e.g., Heliobacter pylori), Aeromonas species (e.g., Aeromonas sobria,Aeromonas hydrophila, Aeromonas caviae), Pleisomonas shigelloides,Yersina enterocolitica, Vibrios species (e.g., Vibrios cholerae, Vibriosparahemolyticus), Klebsiella species, Pseudomonas aeruginosa, andStreptococci. See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds.,Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

Anti-IL-12 antibody compounds, compositions or combinations of thepresent invention can further comprise at least one of any suitableauxiliary, such as, but not limited to, diluent, binder, stabilizer,buffers, salts, lipophilic solvents, preservative, adjuvant or the like.Pharmaceutically acceptable auxiliaries are preferred. Non-limitingexamples of, and methods of preparing such sterile solutions are wellknown in the art, such as, but limited to, Gennaro, Ed., Remington'sPharmaceutical Sciences, 18^(th) Edition, Mack Publishing Co. (Easton,Pa.) 1990. Pharmaceutically acceptable carriers can be routinelyselected that are suitable for the mode of administration, solubilityand/or stability of the anti-IL-12 antibody, fragment or variantcomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include, but are not limited to, proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars, such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin, such ashuman serum albumin (HSA), recombinant human albumin (rHA), gelatin,casein, and the like. Representative amino acid/antibody components,which can also function in a buffering capacity, include alanine,glycine, arginine, betaine, histidine, glutamic acid, aspartic acid,cysteine, lysine, leucine, isoleucine, valine, methionine,phenylalanine, aspartame, and the like. One preferred amino acid isglycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

Anti-IL-12 antibody compositions can also include a buffer or a pHadjusting agent; typically, the buffer is a salt prepared from anorganic acid or base. Representative buffers include organic acid salts,such as salts of citric acid, ascorbic acid, gluconic acid, carbonicacid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris,tromethamine hydrochloride, or phosphate buffers. Preferred buffers foruse in the present compositions are organic acid salts, such as citrate.

Additionally, anti-IL-12 antibody compositions of the invention caninclude polymeric excipients/additives, such as polyvinylpyrrolidones,ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates, such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the anti-IL-12 antibody, portion or variantcompositions according to the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 19^(th) ed.,Williams & Williams, (1995), and in the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), the disclosuresof which are entirely incorporated herein by reference. Preferredcarrier or excipient materials are carbohydrates (e.g., saccharides andalditols) and buffers (e.g., citrate) or polymeric agents. An exemplarycarrier molecule is the mucopolysaccharide, hyaluronic acid, which maybe useful for intraarticular delivery.

Formulations

As noted above, the invention provides for stable formulations, whichpreferably comprise a phosphate buffer with saline or a chosen salt, aswell as preserved solutions and formulations containing a preservativeas well as multi-use preserved formulations suitable for pharmaceuticalor veterinary use, comprising at least one anti-IL-12 antibody in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concentration ormixture can be used as known in the art, such as 0.001-5%, or any rangeor value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one anti-IL-12 antibody with the prescribed buffersand/or preservatives, optionally in an aqueous diluent, wherein saidpackaging material comprises a label that indicates that such solutioncan be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30,36, 40, 48, 54, 60, 66, 72 hours or greater. The invention furthercomprises an article of manufacture, comprising packaging material, afirst vial comprising lyophilized at least one anti-IL-12 antibody, anda second vial comprising an aqueous diluent of prescribed buffer orpreservative, wherein said packaging material comprises a label thatinstructs a patient to reconstitute the at least one anti-IL-12 antibodyin the aqueous diluent to form a solution that can be held over a periodof twenty-four hours or greater.

The at least one anti-IL-12 antibody used in accordance with the presentinvention can be produced by recombinant means, including from mammaliancell or transgenic preparations, or can be purified from otherbiological sources, as described herein or as known in the art.

The range of at least one anti-IL-12 antibody in the product of thepresent invention includes amounts yielding upon reconstitution, if in awet/dry system, concentrations from about 1.0 μg/ml to about 1000 mg/ml,although lower and higher concentrations are operable and are dependenton the intended delivery vehicle, e.g., solution formulations willdiffer from transdermal patch, pulmonary, transmucosal, or osmotic ormicro pump methods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g., isotonicity agents, buffers, antioxidants, andpreservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably, the formulations of the presentinvention have a pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably, sodium phosphate,particularly, phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants, such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators, such asEDTA and EGTA, can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one anti-IL-12 antibody and apreservative selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal or mixtures thereof in anaqueous diluent. Mixing the at least one anti-IL-12 antibody andpreservative in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one anti-IL-12 antibody inbuffered solution is combined with the desired preservative in abuffered solution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that can be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingwater, a preservative and/or excipients, preferably, a phosphate bufferand/or saline and a chosen salt, in an aqueous diluent. Either a singlesolution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus can provide a more convenient treatmentregimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period ranging from immediate to twenty-four hoursor greater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2° C. to about 40° C. and retain the biologically activity of theprotein for extended periods of time, thus allowing a package labelindicating that the solution can be held and/or used over a period of 6,12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent isused, such label can include use up to 1-12 months, one-half, one and ahalf, and/or two years.

The solutions of at least one anti-IL-12 antibody of the invention canbe prepared by a process that comprises mixing at least one antibody inan aqueous diluent. Mixing is carried out using conventional dissolutionand mixing procedures. To prepare a suitable diluent, for example, ameasured amount of at least one antibody in water or buffer is combinedin quantities sufficient to provide the protein and, optionally, apreservative or buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one anti-IL-12antibody that is reconstituted with a second vial containing the aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneanti-IL-12 antibody that is reconstituted with a second vial containingthe aqueous diluent. The clear solution in this case can be up to oneliter or even larger in size, providing a large reservoir from whichsmaller portions of the at least one antibody solution can be retrievedone or multiple times for transfer into smaller vials and provided bythe pharmacy or clinic to their customers and/or patients.

Recognized devices comprising single vial systems include pen-injectordevices for delivery of a solution, such as BD Pens, BD Autojector®,Humaject®, NovoPen®, B-D® Pen, AutoPen®, and OptiPen®, GenotropinPen®,Genotronorm Pen®, Humatro Pen®, Reco-Pen®, Roferon Pen®, Biojector®,Iject®, J-tip Needle-Free Injector®, Intraject®, Medi-Ject®, e.g., asmade or developed by Becton Dickensen (Franklin Lakes, N.J.,www.bectondickenson.com), Disetronic (Burgdorf, Switzerland,www.disetronic.com; Bioject, Portland, Oreg. (www.bioject.com); NationalMedical Products, Weston Medical (Peterborough, UK,www.weston-medical.com), Medi-Ject Corp (Minneapolis, Minn.,www.mediject.com), and similary suitable devices. Recognized devicescomprising a dual vial system include those pen-injector systems forreconstituting a lyophilized drug in a cartridge for delivery of thereconstituted solution, such as the HumatroPen®. Examples of otherdevices suitable include pre-filled syringes, auto-injectors, needlefree injectors, and needle free IV infusion sets.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one anti-IL-12 antibody in theaqueous diluent to form a solution and to use the solution over a periodof 2-24 hours or greater for the two vial, wet/dry, product. For thesingle vial, solution product, the label indicates that such solutioncan be used over a period of 2-24 hours or greater. The presentlyclaimed products are useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one anti-IL-12 antibody and a selectedbuffer, preferably, a phosphate buffer containing saline or a chosensalt. Mixing the at least one anti-IL-12 antibody and buffer in anaqueous diluent is carried out using conventional dissolution and mixingprocedures. To prepare a suitable formulation, for example, a measuredamount of at least one antibody in water or buffer is combined with thedesired buffering agent in water in quantities sufficient to provide theprotein and buffer at the desired concentrations. Variations of thisprocess would be recognized by one of ordinary skill in the art. Forexample, the order the components are added, whether additionaladditives are used, the temperature and pH at which the formulation isprepared, are all factors that can be optimized for the concentrationand means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one anti-IL-12 antibody that is reconstituted with a second vialcontaining a preservative or buffer and excipients in an aqueousdiluent. Either a single solution vial or dual vial requiringreconstitution can be reused multiple times and can suffice for a singleor multiple cycles of patient treatment and thus provides a moreconvenient treatment regimen than currently available.

Other formulations or methods of stablizing the anti-IL-12 antibody mayresult in other than a clear solution of lyophilized powder comprisingthe antibody. Among non-clear solutions are formulations comprisingparticulate suspensions, said particulates being a compositioncontaining the anti-IL-12 antibody in a structure of variable dimensionand known variously as a microsphere, microparticle, nanoparticle,nanosphere, or liposome. Such relatively homogenous, essentiallyspherical, particulate formulations containing an active agent can beformed by contacting an aqueous phase containing the active agent and apolymer and a nonaqueous phase followed by evaporation of the nonaqueousphase to cause the coalescence of particles from the aqueous phase astaught in U.S. Pat. No. 4,589,330. Porous microparticles can be preparedusing a first phase containing active agent and a polymer dispersed in acontinuous solvent and removing said solvent from the suspension byfreeze-drying or dilution-extraction-precipitation as taught in U.S.Pat. No. 4,818,542. Preferred polymers for such preparations are naturalor synthetic copolymers or polymers selected from the group consistingof gleatin agar, starch, arabinogalactan, albumin, collagen,polyglycolic acid, polylactic aced, glycolide-L(−) lactidepoly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lactic acid),poly(epsilon-caprolactone-CO-glycolic acid), poly(β-hydroxy butyricacid), polyethylene oxide, polyethylene, poly(alkyl-2-cyanoacrylate),poly(hydroxyethyl methacrylate), polyamides, poly(amino acids),poly(2-hydroxyethyl DL-aspartamide), poly(ester urea),poly(L-phenylalanine/ethylene glycol/1,6-diisocyanatohexane) andpoly(methyl methacrylate). Particularly preferred polymers arepolyesters, such as polyglycolic acid, polylactic aced, glycolide-L(−)lactide poly(episilon-caprolactone, poly(epsilon-caprolactone-CO-lacticacid), and poly(epsilon-caprolactone-CO-glycolic acid. Solvents usefulfor dissolving the polymer and/or the active include: water,hexafluoroisopropanol, methylenechloride, tetrahydrofuran, hexane,benzene, or hexafluoroacetone sesquihydrate. The process of dispersingthe active containing phase with a second phase may include pressureforcing said first phase through an orifice in a nozzle to affectdroplet formation.

Dry powder formulations may result from processes other thanlyophilization, such as by spray drying or solvent extraction byevaporation or by precipitation of a crystalline composition followed byone or more steps to remove aqueous or nonaqueous solvent. Preparationof a spray-dried antibody preparation is taught in U.S. Pat. No.6,019,968. The antibody-based dry powder compositions may be produced byspray drying solutions or slurries of the antibody and, optionally,excipients, in a solvent under conditions to provide a respirable drypowder. Solvents may include polar compounds, such as water and ethanol,which may be readily dried. Antibody stability may be enhanced byperforming the spray drying procedures in the absence of oxygen, such asunder a nitrogen blanket or by using nitrogen as the drying gas. Anotherrelatively dry formulation is a dispersion of a plurality of perforatedmicrostructures dispersed in a suspension medium that typicallycomprises a hydrofluoroalkane propellant as taught in WO 9916419. Thestabilized dispersions may be administered to the lung of a patientusing a metered dose inhaler. Equipment useful in the commercialmanufacture of spray dried medicaments are manufactured by Buchi Ltd. orNiro Corp.

At least one anti-IL-12 antibody in either the stable or preservedformulations or solutions described herein, can be administered to apatient in accordance with the present invention via a variety ofdelivery methods including SC or IM injection; transdermal, pulmonary,transmucosal, implant, osmotic pump, cartridge, micro pump, or othermeans appreciated by the skilled artisan, as well-known in the art.

Therapeutic Applications

The present invention also provides a method for modulating or treatingat least one IL-12 related disease, in a cell, tissue, organ, animal, orpatient, as known in the art or as described herein, using at least oneIL-12 antibody of the present invention, e.g., administering orcontacting the cell, tissue, organ, animal, or patient with atherapeutic effective amount of IL-12 antibody. The present inventionalso provides a method for modulating or treating at least one IL-12related disease, in a cell, tissue, organ, animal, or patient including,but not limited to, at least one of obesity, an immune related disease,a cardiovascular disease, an infectious disease, a malignant disease ora neurologic disease.

The present invention also provides a method for modulating or treatingat least one IL-12 related immune related disease, in a cell, tissue,organ, animal, or patient including, but not limited to, at least one ofrheumatoid arthritis, juvenile rheumatoid arthritis, systemic onsetjuvenile rheumatoid arthritis, psoriatic arthritis, ankylosingspondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis,osteolysis, aseptic loosening of orthopedic implants, inflammatory boweldisease, ulcerative colitis, systemic lupus erythematosus,antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis,idiopathic pulmonary fibrosis, systemic vasculitis/wegener'sgranulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures,allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergiccontact dermatitis, allergic conjunctivitis, hypersensitivitypneumonitis, transplants, organ transplant rejection, graft-versus-hostdisease, systemic inflammatory response syndrome, sepsis syndrome, grampositive sepsis, gram negative sepsis, culture negative sepsis, fungalsepsis, neutropenic fever, urosepsis, meningococcemia,trauma/hemorrhage, burns, ionizing radiation exposure, acutepancreatitis, adult respiratory distress syndrome, rheumatoid arthritis,alcohol-induced hepatitis, chronic inflammatory pathologies,sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis,atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever,perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria,systemic anaphalaxis, dermatitis, pernicious anemia, hemolyticdisesease, thrombocytopenia, graft rejection of any organ or tissue,kidney translplant rejection, heart transplant rejection, livertransplant rejection, pancreas transplant rejection, lung transplantrejection, bone marrow transplant (BMT) rejection, skin allograftrejection, cartilage transplant rejection, bone graft rejection, smallbowel transplant rejection, fetal thymus implant rejection, parathyroidtransplant rejection, xenograft rejection of any organ or tissue,allograft rejection, anti-receptor hypersensitivity reactions, Gravesdisease, Raynaud's disease, type B insulin-resistant diabetes, asthma,myasthenia gravis, antibody-meditated cytotoxicity, type IIIhypersensitivity reactions, POEMS syndrome (polyneuropathy,organomegaly, endocrinopathy, monoclonal gammopathy, and skin changessyndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonalgammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus,scleroderma, mixed connective tissue disease, idiopathic Addison'sdisease, diabetes mellitus, chronic active hepatitis, primary billiarycirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IVhypersensitivity, contact dermatitis, hypersensitivity pneumonitis,allograft rejection, granulomas due to intracellular organisms, drugsensitivity, metabolic/idiopathic, Wilson's disease, hemachromatosis,alpha-1-antitrypsin deficiency, diabetic retinopathy, hashimoto'sthyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axisevaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis,cachexia, cystic fibrosis, neonatal chronic lung disease, chronicobstructive pulmonary disease (COPD), familial hematophagocyticlymphohistiocytosis, dermatologic conditions, psoriasis, alopecia,nephrotic syndrome, nephritis, glomerular nephritis, acute renalfailure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy,anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy(e.g., including but not limited to, asthenia, anemia, cachexia, and thelike), chronic salicylate intoxication, and the like. See, e.g., theMerck Manual, 12th-17th Editions, Merck & Company, Rahway, N.J. (1972,1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al.,eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000),each entirely incorporated by reference.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis,restenosis, diabetic ateriosclerotic disease, hypertension, arterialhypertension, renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), post perfusion syndrome,cardiopulmonary bypass inflammation response, chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aordic andperipheral aneuryisms, aortic dissection, inflammation of the aorta,occlusion of the abdominal aorta and its branches, peripheral vasculardisorders, occlusive arterial disorders, peripheral atherloscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy.

The present invention also provides a method for modulating or treatingat least one IL-12 related infectious disease in a cell, tissue, organ,animal or patient, including, but not limited to, at least one of: acuteor chronic bacterial infection, acute and chronic parasitic orinfectious processes, including bacterial, viral and fungal infections,HIV infection/HIV neuropathy, meningitis, hepatitis (e.g., A, B or C, orthe like), septic arthritis, peritonitis, pneumonia, epiglottitis, E.coli 0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenicpurpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,toxic shock syndrome, streptococcal myositis, gas gangrene,mycobacterium tuberculosis, mycobacterium avium intracellulare,pneumocystis carinii pneumonia, pelvic inflammatory disease,orchitis/epidydimitis, legionella, lyme disease, influenza a,epstein-barr virus, viral-associated hemaphagocytic syndrome, viralencephalitis/aseptic meningitis, and the like.

The present invention also provides a method for modulating or treatingat least one IL-12 related malignant disease in a cell, tissue, organ,animal or patient, including, but not limited to, at least one of:leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), acutelymphocytic leukemia, B-cell, T-cell or FAB ALL, acute myeloid leukemia(AML), acute myelogenous leukemia, chromic myelocytic leukemia (CML),chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplasticsyndrome (MDS), a lymphoma, Hodgkin's disease, a malignamt lymphoma,non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi'ssarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngealcarcinoma, malignant histiocytosis, paraneoplasticsyndrome/hypercalcemia of malignancy, solid tumors, bladder cancer,breast cancer, colorectal cancer, endometiral cancer, head cancer, neckcancer, hereditary nonpolyposis cancer, Hodgkin's lymphoma, livercancer, lung cancer, non-small cell lung cancer, ovarian cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, testicularcancer, adenocarcinomas, sarcomas, malignant melanoma, hemangioma,metastatic disease, cancer related bone resorption, cancer related bonepain, and the like.

The present invention also provides a method for modulating or treatingat least one IL-12 related neurologic disease in a cell, tissue, organ,animal or patient, including, but not limited to, at least one of:neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders, suchas lesions of the corticospinal system; disorders of the basal ganglia;hyperkinetic movement disorders, such as Huntington's Chorea and senilechorea; drug-induced movement disorders, such as those induced by drugswhich block CNS dopamine receptors; hypokinetic movement disorders, suchas Parkinson's disease; Progressive supranucleo Palsy; structurallesions of the cerebellum; spinocerebellar degenerations, such as spinalataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiplesystems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, andMachado-Joseph); systemic disorders (Refsum's disease,abetalipoprotemia, ataxia, telangiectasia, and mitochondrialmulti-system disorder); demyelinating core disorders, such as multiplesclerosis, acute transverse myelitis; and disorders of the motor unit,such as neurogenic muscular atrophies (anterior horn cell degeneration,such as amyotrophic lateral sclerosis, infantile spinal muscular atrophyand juvenile spinal muscular atrophy); Alzheimer's disease; Down'sSyndrome in middle age; Diffuse Lewy body disease; Senile Dementia ofLewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism;Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis,Hallerrorden-Spatz disease; Dementia pugilistica; neurotraumatic injury(e.g., spinal cord injury, brain injury, concussion, repetitiveconcussion); pain; inflammatory pain; autism; depression; stroke;cognitive disorders; epilepsy; and the like. Such a method canoptionally comprise administering an effective amount of a compositionor pharmaceutical composition comprising at least one TNF antibody orspecified portion or variant to a cell, tissue, organ, animal or patientin need of such modulation, treatment or therapy. See, e.g., the MerckManual, 16^(th) Edition, Merck & Company, Rahway, N.J. (1992).

The present invention also provides a method for modulating or treatingat least one IL-12 related wound, trauma or tissue injury or relatedchronic condition, in a cell, tissue, organ, animal or patient,including, but not limited to, at least one of: bodily injury or atrauma associated with oral surgery including periodontal surgery, toothextraction(s), endodontic treatment, insertion of tooth implants,application and use of tooth prosthesis; or wherein the wound isselected from the group consisting of aseptic wounds, contused wounds,incised wounds, lacerated wounds, non-penetrating wounds, open wounds,penetrating wounds, perforating wounds, puncture wounds, septic wounds,infarctions and subcutaneous wounds; or wherein the wound is selectedfrom the group consisting of ischemic ulcers, pressure sores, fistulae,severe bites, thermal burns and donor site wounds; or wherein the woundis anaphthous wound, a traumatic wound or a herpes associated wound.

Wounds and/or ulcers are normally found protruding from the skin or on amucosal surface or as a result of an infarction in an organ (“stroke”).A wound may be a result of a soft tissue defect or a lesion or of anunderlying condition. In the present context, the term “skin” relates tothe outermost surface of the body of an animal, including a human, andembraces intact or almost intact skin as well as an injured skinsurface. The term “mucosa” relates to undamaged or damaged mucosa of ananimal, such as a human, and may be the oral, buccal, aural, nasal,lung, eye, gastrointestinal, vaginal, or rectal mucosa.

In the present context the term “wound” denotes a bodily injury withdisruption of the normal integrity of tissue structures. The term isalso intended to encompass the terms “sore,” “lesion,” “necrosis,” and“ulcer.” Normally, the term “sore” is a popular term for almost anylesion of the skin or mucous membranes and the term “ulcer” is a localdefect, or excavation, of the surface of an organ or tissue, which isproduced by the sloughing of necrotic tissue. Lesion generally relatesto any tissue defect. Necrosis is related to dead tissue resulting frominfection, injury, inflammation or infarctions.

The term “wound” used in the present context denotes any wound (seebelow for a classification of wounds) and at any particular stage in thehealing process, including the stage before any healing has initiated oreven before a specific wound like a surgical incision is made(prophylactic treatment). Examples of wounds which can be preventedand/or treated in accordance with the present invention are, e.g.,aseptic wounds, contused wounds, incised wounds, lacerated wounds,non-penetrating wounds (i.e., wounds in which there is no disruption ofthe skin but there is injury to underlying structures), open wounds,penetrating wounds, perforating wounds, puncture wounds, septic wounds,subcutaneous wounds, etc. Examples of sores are bed sores, canker sores,chrome sores, cold sores, pressure sores, etc. Examples of ulcers are,e.g., a peptic ulcer, duodenal ulcer, gastric ulcer, gouty ulcer,diabetic ulcer, hypertensive ischemic ulcer, stasis ulcer, ulcus cruris(venous ulcer), sublingual ulcer, submucous ulcer, symptomatic ulcer,trophic ulcer, tropical ulcer, and veneral ulcer, e.g., caused bygonorrhoea (including urethritis, endocervicitis and proctitis).Conditions related to wounds or sores which may be successfully treatedaccording to the invention are burns, anthrax, tetanus, gas gangrene,scalatina, erysipelas, sycosis barbae, folliculitis, impetigocontagiosa, or impetigo bullosa, etc. There is often a certain overlapbetween the use of the terms “wound” and “ulcer” and “wound” and “sore”and, furthermore, the terms are often used at random. Therefore, asmentioned above, in the present context the term “wound” encompasses theterms “ulcer,” “lesion,” “sore” and “infarction,” and the terms areindiscriminately used unless otherwise indicated.

The kinds of wounds to be treated according to the invention includealso (i) general wounds, such as, e.g., surgical, traumatic, infectious,ischemic, thermal, chemical and bullous wounds; (ii) wounds specific forthe oral cavity, such as, e.g., post-extraction wounds, endodonticwounds especially in connection with treatment of cysts and abscesses,ulcers and lesions of bacterial, viral or autoimmunological origin,mechanical, chemical, thermal, infectious and lichenoid wounds; herpesulcers, stomatitis aphthosa, acute necrotising ulcerative gingivitis andburning mouth syndrome are specific examples; and (iii) wounds on theskin, such as, e.g., neoplasm, burns (e.g. chemical, thermal), lesions(bacterial, viral, autoimmunological), bites and surgical incisions.Another way of classifying wounds is as (i) small tissue loss due tosurgical incisions, minor abrasions and minor bites, or as (ii)significant tissue loss. The latter group includes ischemic ulcers,pressure sores, fistulae, lacerations, severe bites, thermal burns anddonor site wounds (in soft and hard tissues) and infarctions.

Other wounds that are of importance in connection with the presentinvention are wounds like ischemic ulcers, pressure sores, fistulae,severe bites, thermal burns and donor site wounds. Ischemic ulcers andpressure sores are wounds which normally only heal very slowly andespecially in such cases, an improved and more rapid healing process isof course of great importance for the patient. Furthermore, the costsinvolved in the treatment of patients suffering from such wounds aremarkedly reduced when the healing is improved and takes place morerapidly.

Donor site wounds are wounds which, e.g., occur in connection withremoval of hard tissue from one part of the body to another part of thebody, e.g., in connection with transplantation. The wounds resultingfrom such operations are very painful and an improved healing istherefore most valuable. The term “skin” is used in a very broad senseembracing the epidermal layer of the skin and—in those cases where theskin surface is more or less injured—also the dermal layer of the skin.Apart from the stratum corneum, the epidermal layer of the skin is theouter (epithelial) layer and the deeper connective tissue layer of theskin is called the dermis.

The present invention also provides a method for modulating or treatingCrohn's disease, psoriasis, and multiple sclerosis, among the otherdiseases listed above as IL-12 related, in a cell, tissue, organ,animal, or patient including, but not limited to, at least one of immunerelated disease, cardiovascular disease, infectious, malignant and/orneurologic disease. Such a method can optionally comprise administeringan effective amount of at least one composition or pharmaceuticalcomposition comprising at least one anti-IL-12 antibody to a cell,tissue, organ, animal or patient in need of such modulation, treatmentor therapy.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one anti-IL-12 antibody to a cell, tissue, organ,animal or patient in need of such modulation, treatment or therapy. Sucha method can optionally further comprise co-administration orcombination therapy for treating such diseases or disorders, wherein theadministering of said at least one anti-IL-12 antibody, specifiedportion or variant thereof, further comprises administering, beforeconcurrently, and/or after, at least one selected from at least one TNFantagonist (e.g., but not limited to, a TNF chemical or proteinantagonist, TNF monoclonal or polyclonal antibody or fragment, a solubleTNF receptor (e.g., p55, p70 or p85) or fragment, fusion polypeptidesthereof, or a small molecule TNF antagonist, e.g., TNF binding protein Ior II (TBP-1 or TBP-II), nerelimonmab, infliximab, eternacept (Enbrel™),adalimulab (Humira™), CDP-571, CDP-870, afelimomab, lenercept, and thelike), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose,azathioprine, gold sodium thiomalate, hydroxychloroquine sulfate,leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anesthetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a fluororquinolone, a macrolide, apenicillin, a sulfonamide, a tetracycline, another antimicrobial), anantipsoriatic, a corticosteriod, an anabolic steroid, a diabetes relatedagent, a mineral, a nutritional, a thyroid agent, a vitamin, a calciumrelated hormone, an antidiarrheal, an antitussive, an antiemetic, anantiulcer, a laxative, an anticoagulant, an erythropoietin (e.g.,epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim(GM-CSF, Leukine), an immunization, an immunoglobulin, animmunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), agrowth hormone, a hormone replacement drug, an estrogen receptormodulator, a mydriatic, a cycloplegic, an alkylating agent, anantimetabolite, a mitotic inhibitor, a radiopharmaceutical, anantidepressant, antimanic agent, an antipsychotic, an anxiolytic, ahypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Suitable dosages arewell known in the art. See, e.g., Wells et al., eds., PharmacotherapyHandbook, 2^(nd) Edition, Appleton and Lange, Stamford, Conn. (2000);PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition,Tarascon Publishing, Loma Linda, Calif. (2000); Nursing 2001 Handbook ofDrugs, 21^(st) edition, Springhouse Corp., Springhouse, Pa., 2001;Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang,Prentice-Hall, Inc, Upper Saddle River, N.J. each of which referencesare entirely incorporated herein by reference.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one antibody, specified portion and variantthereof, of the present invention), include, but are not limited to,anti-TNF antibodies (e.g., at least one TNF antagonist as definedabove), antigen-binding fragments thereof, and receptor molecules whichbind specifically to TNF; compounds which prevent and/or inhibit TNFsynthesis, TNF release or its action on target cells, such asthalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifyllineand rolipram), A2b adenosine receptor agonists and A2b adenosinereceptor enhancers; compounds which prevent and/or inhibit TNF receptorsignalling, such as mitogen activated protein (MAP) kinase inhibitors;compounds which block and/or inhibit membrane TNF cleavage, such asmetalloproteinase inhibitors; compounds which block and/or inhibit TNFactivity, such as angiotensin converting enzyme (ACE) inhibitors (e.g.,captopril); and compounds which block and/or inhibit TNF productionand/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/or,preferably, in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

An example of a TNF antibody or antagonist is the chimeric antibody cA2.Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

TNF Receptor Molecules

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFα with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). An example of such a TNFimmunoreceptor fusion molecule is TNF receptor/IgG fusion protein. TNFimmunoreceptor fusion molecules and methods for their production havebeen described in the art (Lesslauer et al., Eur. J. Immunol.21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Peppel et al., J. Exp. Med. 174:1483-1489 (1991);Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219 (1994); Butler etal., Cytokine 6(6):616-623 (1994); Baker et al., Eur. J. Immunol.24:2040-2048 (1994); Beutler et al., U.S. Pat. No. 5,447,851; and U.S.application Ser. No. 08/442,133 (filed May 16, 1995), each of whichreferences are entirely incorporated herein by reference). Methods forproducing immunoreceptor fusion molecules can also be found in Capon etal., U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538; andCapon et al., Nature 337:525-531 (1989), which references are entirelyincorporated herein by reference.

Cytokines include any known cytokine. See, e.g., CopewithCytokines.com.Cytokine antagonists include, but are not limited to, any antibody,fragment or mimetic, any soluble receptor, fragment or mimetic, anysmall molecule antagonist, or any combination thereof.

Therapeutic Treatments

Any method of the present invention can comprise a method for treatingan IL-12 mediated disorder, comprising administering an effective amountof a composition or pharmaceutical composition comprising at least oneanti-IL-12 antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy. Such a method can optionallyfurther comprise co-administration or combination therapy for treatingsuch diseases or disorders, wherein the administering of said at leastone anti-IL-12 antibody, specified portion or variant thereof, furthercomprises administering before, concurrently, and/or after, at least oneselected from an anti-infective drug, a cardiovascular (CV) system drug,a central nervous system (CNS) drug, an autonomic nervous system (ANS)drug, a respiratory tract drug, a gastrointestinal (GI) tract drug, ahormonal drug, a drug for fluid or electrolyte balance, a hematologicdrug, an antineoplastic, an immunomodulation drug, an ophthalmic, oticor nasal drug, a topical drug, a nutritional drug or the like, at leastone TNF antagonist (e.g., but not limited to a TNF antibody or fragment,a soluble TNF receptor or fragment, fusion proteins thereof, or a smallmolecule TNF antagonist), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid anti-inflammatory drug(NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, aneuromuscular blocker, an antimicrobial (e.g., aminoglycoside, anantifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin,a fluororquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropoietin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Such drugs are well known in the art, includingformulations, indications, dosing and administration for each presentedherein (see., e.g., Nursing 2001 Handbook of Drugs, 21^(st) edition,Springhouse Corp., Springhouse, Pa., 2001; Health Professional's DrugGuide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, UpperSaddle River, N.J.; Pharmcotherapy Handbook, Wells et al., ed., Appleton& Lange, Stamford, Conn., each entirely incorporated herein byreference).

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one anti-IL-12antibody composition that total, on average, a range from at least about0.01 to 500 milligrams of at least one anti-IL-12 antibody per kilogramof patient per dose, and, preferably, from at least about 0.1 to 100milligrams antibody/kilogram of patient per single or multipleadministration, depending upon the specific activity of the active agentcontained in the composition. Alternatively, the effective serumconcentration can comprise 0.1-5000 μg/ml serum concentration per singleor multiple administration. Suitable dosages are known to medicalpractitioners and will, of course, depend upon the particular diseasestate, specific activity of the composition being administered, and theparticular patient undergoing treatment. In some instances, to achievethe desired therapeutic amount, it can be necessary to provide forrepeated administration, i.e., repeated individual administrations of aparticular monitored or metered dose, where the individualadministrations are repeated until the desired daily dose or effect isachieved.

Preferred doses can optionally include 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and/or 100-500mg/kg/administration, or any range, value or fraction thereof, or toachieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9,2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5,6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11,11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0,5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14,14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9,19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500,and/or 5000 μg/ml serum concentration per single or multipleadministration, or any range, value or fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and, preferably, 0.1 to10 milligrams per kilogram per administration or in sustained releaseform is effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one antibody ofthe present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or, alternatively oradditionally, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, or 52, or, alternatively or additionally, at least oneof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 years, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.001 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-99.999% by weight based on the total weight of thecomposition.

For parenteral administration, the antibody can be formulated as asolution, suspension, emulsion, particle, powder, or lyophilized powderin association, or separately provided, with a pharmaceuticallyacceptable parenteral vehicle. Examples of such vehicles are water,saline, Ringer's solution, dextrose solution, and 1-10% human serumalbumin. Liposomes and nonaqueous vehicles, such as fixed oils, can alsobe used. The vehicle or lyophilized powder can contain additives thatmaintain isotonicity (e.g., sodium chloride, mannitol) and chemicalstability (e.g., buffers and preservatives). The formulation issterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Alternative Administration

Many known and developed modes can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one anti-IL-12 antibody according to the present invention. Whilepulmonary administration is used in the following description, othermodes of administration can be used according to the present inventionwith suitable results. IL-12 antibodies of the present invention can bedelivered in a carrier, as a solution, emulsion, colloid, or suspension,or as a dry powder, using any of a variety of devices and methodssuitable for administration by inhalation or other modes described herewithin or known in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols, such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent, such as aqueous solution, asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least oneanti-IL-12 antibody by parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermalmeans. At least one anti-IL-12 antibody composition can be prepared foruse for parenteral (subcutaneous, intramuscular or intravenous) or anyother administration particularly in the form of liquid solutions orsuspensions; for use in vaginal or rectal administration particularly insemisolid forms, such as, but not limited to, creams and suppositories;for buccal, or sublingual administration, such as, but not limited to,in the form of tablets or capsules; or intranasally, such as, but notlimited to, the form of powders, nasal drops or aerosols or certainagents; or transdermally, such as not limited to a gel, ointment,lotion, suspension or patch delivery system with chemical enhancers suchas dimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement;” Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathways,such as electroporation, or to increase the mobility of charged drugsthrough the skin, such as iontophoresis, or application of ultrasound,such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably, at least one anti-IL-12antibody composition is delivered in a particle size effective forreaching the lower airways of the lung or sinuses. According to theinvention, at least one anti-IL-12 antibody can be delivered by any of avariety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of antibodies are alsoknown in the art. All such devices can use formulations suitable for theadministration for the dispensing of antibody in an aerosol. Suchaerosols can be comprised of either solutions (both aqueous and nonaqueous) or solid particles.

Metered dose inhalers like the Ventolin® metered dose inhaler, typicallyuse a propellent gas and require actuation during inspiration (See,e.g., WO 94/16970, WO 98/35888). Dry powder inhalers like Turbuhaler™(Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ inhaler (Dura),devices marketed by Inhale Therapeutics, and the Spinhaler® powderinhaler (Fisons), use breath-actuation of a mixed powder (U.S. Pat. No.4,668,218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura,U.S. Pat. No. 5,458,135 Inhale, WO 94/06498 Fisons, entirelyincorporated herein by reference). Nebulizers like AERx™ Aradigm, theUltravent® nebulizer (Mallinckrodt), and the Acorn II® nebulizer(Marquest Medical Products) (U.S. Pat. No. 5,404,871 Aradigm, WO97/22376), the above references entirely incorporated herein byreference, produce aerosols from solutions, while metered dose inhalers,dry powder inhalers, etc. generate small particle aerosols. Thesespecific examples of commercially available inhalation devices areintended to be a representative of specific devices suitable for thepractice of this invention, and are not intended as limiting the scopeof the invention.

Preferably, a composition comprising at least one anti-IL-12 antibody isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone antibody of the present invention. For example, delivery by theinhalation device is advantageously reliable, reproducible, andaccurate. The inhalation device can optionally deliver small dryparticles, e.g., less than about 10 μm, preferably about 1-5 μm, forgood respirability.

Administration of IL-12 Antibody Compositions as a Spray

A spray including IL-12 antibody composition can be produced by forcinga suspension or solution of at least one anti-IL-12 antibody through anozzle under pressure. The nozzle size and configuration, the appliedpressure, and the liquid feed rate can be chosen to achieve the desiredoutput and particle size. An electrospray can be produced, for example,by an electric field in connection with a capillary or nozzle feed.Advantageously, particles of at least one anti-IL-12 antibodycomposition delivered by a sprayer have a particle size less than about10 μm, preferably, in the range of about 1 μm to about 5 μm, and, mostpreferably, about 2 μm to about 3 μm.

Formulations of at least one anti-IL-12 antibody composition suitablefor use with a sprayer typically include antibody composition in anaqueous solution at a concentration of about 0.1 mg to about 100 mg ofat least one anti-IL-12 antibody composition per ml of solution ormg/gm, or any range or value therein, e.g., but not limited to, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/ml or mg/gm. Theformulation can include agents, such as an excipient, a buffer, anisotonicity agent, a preservative, a surfactant, and, preferably, zinc.The formulation can also include an excipient or agent for stabilizationof the antibody composition, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating antibodycompositions include albumin, protamine, or the like. Typicalcarbohydrates useful in formulating antibody compositions includesucrose, mannitol, lactose, trehalose, glucose, or the like. Theantibody composition formulation can also include a surfactant, whichcan reduce or prevent surface-induced aggregation of the antibodycomposition caused by atomization of the solution in forming an aerosol.Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitol fatty acid esters. Amounts will generally range between 0.001and 14% by weight of the formulation. Especially preferred surfactantsfor purposes of this invention are polyoxyethylene sorbitan monooleate,polysorbate 80, polysorbate 20, or the like. Additional agents known inthe art for formulation of a protein, such as IL-12 antibodies, orspecified portions or variants, can also be included in the formulation.

Administration of IL-12 Antibody Compositions by a Nebulizer

Antibody compositions of the invention can be administered by anebulizer, such as a jet nebulizer or an ultrasonic nebulizer.Typically, in a jet nebulizer, a compressed air source is used to createa high-velocity air jet through an orifice. As the gas expands beyondthe nozzle, a low-pressure region is created, which draws a solution ofantibody composition through a capillary tube connected to a liquidreservoir. The liquid stream from the capillary tube is sheared intounstable filaments and droplets as it exits the tube, creating theaerosol. A range of configurations, flow rates, and baffle types can beemployed to achieve the desired performance characteristics from a givenjet nebulizer. In an ultrasonic nebulizer, high-frequency electricalenergy is used to create vibrational, mechanical energy, typicallyemploying a piezoelectric transducer. This energy is transmitted to theformulation of antibody composition either directly or through acoupling fluid, creating an aerosol including the antibody composition.Advantageously, particles of antibody composition delivered by anebulizer have a particle size less than about 10 μm, preferably, in therange of about 1 μm to about 5 μm, and, most preferably, about 2 μm toabout 3 μm.

Formulations of at least one anti-IL-12 antibody suitable for use with anebulizer, either jet or ultrasonic, typically include a concentrationof about 0.1 mg to about 100 mg of at least one anti-IL-12 antibodyprotein per ml of solution. The formulation can include agents, such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one anti-IL-12antibody composition, such as a buffer, a reducing agent, a bulkprotein, or a carbohydrate. Bulk proteins useful in formulating at leastone anti-IL-12 antibody composition include albumin, protamine, or thelike. Typical carbohydrates useful in formulating at least oneanti-IL-12 antibody include sucrose, mannitol, lactose, trehalose,glucose, or the like. The at least one anti-IL-12 antibody formulationcan also include a surfactant, which can reduce or preventsurface-induced aggregation of the at least one anti-IL-12 antibodycaused by atomization of the solution in forming an aerosol. Variousconventional surfactants can be employed, such as polyoxyethylene fattyacid esters and alcohols, and polyoxyethylene sorbital fatty acidesters. Amounts will generally range between 0.001 and 4% by weight ofthe formulation. Especially preferred surfactants for purposes of thisinvention are polyoxyethylene sorbitan mono-oleate, polysorbate 80,polysorbate 20, or the like. Additional agents known in the art forformulation of a protein, such as antibody protein, can also be includedin the formulation.

Administration of IL-12 Antibody Compositions by a Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one anti-IL-12antibody, and any excipients or other additives are contained in acanister as a mixture including a liquefied compressed gas. Actuation ofthe metering valve releases the mixture as an aerosol, preferablycontaining particles in the size range of less than about 10 μm,preferably, about 1 μm to about 5 μm, and, most preferably, about 2 μmto about 3 μm. The desired aerosol particle size can be obtained byemploying a formulation of antibody composition produced by variousmethods known to those of skill in the art, including jet-milling, spraydrying, critical point condensation, or the like. Preferred metered doseinhalers include those manufactured by 3M or Glaxo and employing ahydrofluorocarbon propellant. Formulations of at least one anti-IL-12antibody for use with a metered-dose inhaler device will generallyinclude a finely divided powder containing at least one anti-IL-12antibody as a suspension in a non-aqueous medium, for example, suspendedin a propellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the like.Preferably, the propellant is a hydrofluorocarbon. The surfactant can bechosen to stabilize the at least one anti-IL-12 antibody as a suspensionin the propellant, to protect the active agent against chemicaldegradation, and the like. Suitable surfactants include sorbitantrioleate, soya lecithin, oleic acid, or the like. In some cases,solution aerosols are preferred using solvents, such as ethanol.Additional agents known in the art for formulation of a protein can alsobe included in the formulation. One of ordinary skill in the art willrecognize that the methods of the current invention can be achieved bypulmonary administration of at least one anti-IL-12 antibody compositionvia devices not described herein.

Oral Formulations and Administration

Formulations for oral administration rely on the co-administration ofadjuvants (e.g., resorcinols and nonionic surfactants, such aspolyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) toincrease artificially the permeability of the intestinal walls, as wellas the co-administration of enzymatic inhibitors (e.g., pancreatictrypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) toinhibit enzymatic degradation. Formulations for delivery of hydrophilicagents including proteins and antibodies and a combination of at leasttwo surfactants intended for oral, buccal, mucosal, nasal, pulmonary,vaginal transmembrane, or rectal administration are taught in U.S. Pat.No. 6,309,663. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant, such as magnesium stearate, paraben, preserving agent,such as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations can contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 and used to deliver biologically active agents orally areknown in the art.

Mucosal Formulations and Administration

A formulation for orally administering a bioactive agent encapsulated inone or more biocompatible polymer or copolymer excipients, preferably, abiodegradable polymer or copolymer, affording microcapsules which due tothe proper size of the resultant microcapsules results in the agentreaching and being taken up by the folliculi lymphatic aggregati,otherwise known as the “Peyer's patch,” or “GALT” of the animal withoutloss of effectiveness due to the agent having passed through thegastrointestinal tract. Similar folliculi lymphatic aggregati can befound in the bronchei tubes (BALT) and the large intestine. Theabove-described tissues are referred to in general as mucosallyassociated lymphoreticular tissues (MALT). For absorption throughmucosal surfaces, compositions and methods of administering at least oneanti-IL-12 antibody include an emulsion comprising a plurality ofsubmicron particles, a mucoadhesive macromolecule, a bioactive peptide,and an aqueous continuous phase, which promotes absorption throughmucosal surfaces by achieving mucoadhesion of the emulsion particles(U.S. Pat. No. 5,514,670). Mucous surfaces suitable for application ofthe emulsions of the present invention can include corneal,conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic,intestinal, and rectal routes of administration. Formulations forvaginal or rectal administration, e.g., suppositories, can contain asexcipients, for example, polyalkyleneglycols, vaseline, cocoa butter,and the like. Formulations for intranasal administration can be solidand contain as excipients, for example, lactose or can be aqueous oroily solutions of nasal drops. For buccal administration, excipientsinclude sugars, calcium stearate, magnesium stearate, pregelinatinedstarch, and the like (U.S. Pat. No. 5,849,695).

Transdermal Formulations and Administration

For transdermal administration, the at least one anti-IL-12 antibody isencapsulated in a delivery device, such as a liposome or polymericnanoparticles, microparticle, microcapsule, or microspheres (referred tocollectively as microparticles unless otherwise stated). A number ofsuitable devices are known, including microparticles made of syntheticpolymers, such as polyhydroxy acids, such as polylactic acid,polyglycolic acid and copolymers thereof, polyorthoesters,polyanhydrides, and polyphosphazenes, and natural polymers, such ascollagen, polyamino acids, albumin and other proteins, alginate andother polysaccharides, and combinations thereof (U.S. Pat. No.5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid, such as phosphoric acid, sulfuric acid, citric acid, tartaricacid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and thelike; (b) a salt with a polyvalent metal cation, such as zinc, calcium,bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmiumand the like, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b), e.g., a zinc tannate salt. Additionally, the compounds ofthe present invention or, preferably, a relatively insoluble salt, suchas those just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g., sesame oil, suitable forinjection. Particularly preferred salts are zinc salts, zinc tannatesalts, pamoate salts, and the like. Another type of slow release depotformulation for injection would contain the compound or salt dispersedfor encapsulation in a slow degrading, non-toxic, non-antigenic polymer,such as a polylactic acid/polyglycolic acid polymer for example asdescribed in U.S. Pat. No. 3,773,919. The compounds or, preferably,relatively insoluble salts, such as those described above, can also beformulated in cholesterol matrix silastic pellets, particularly for usein animals. Additional slow release, depot or implant formulations,e.g., gas or liquid liposomes, are known in the literature (U.S. Pat.No. 5,770,222 and “Sustained and Controlled Release Drug DeliverySystems”, J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1 Cloning and Expression of IL-12 Antibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, theantibody coding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors, such as pIRES1neo, pRetro-Off,pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1(+/−), pcDNA/Zeo (+/−) or pcDNA3.1/Hygro (+/−) (Invitrogen), PSVL andPMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells. Alternatively, the gene can beexpressed in stable cell lines that contain the gene integrated into achromosome. The co-transfection with a selectable marker, such as dhfr,gpt, neomycin, or hygromycin, allows the identification and isolation ofthe transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded antibody. The DHFR (dihydrofolate reductase) marker isuseful to develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J.227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of antibodies.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition to the 3′ intron,the polyadenylation and termination signal of the rat preproinsulingene.

Cloning and Expression in CHO Cells.

The vector pC4 is used for the expression of IL-12 antibody. Plasmid pC4is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Theplasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (e.g., alpha minus MEM, LifeTechnologies, Gaithersburg, Md.) supplemented with the chemotherapeuticagent methotrexate. The amplification of the DHFR genes in cellsresistant to methotrexate (MTX) has been well documented (see, e.g., F.W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C.Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M.A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

High efficiency promoters other than the strong promoter of the longterminal repeat (LTR) of the Rous Sarcoma Virus can also be used for theexpression, e.g., the human b-actin promoter, the SV40 early or latepromoters or the long terminal repeats from other retroviruses, e.g.,HIV and HTLVI. Clontech's Tet-Off and Tet-On gene expression systems andsimilar systems can be used to express the IL-12 in a regulated way inmammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA89: 5547-5551 (1992)). For the polyadenylation of the mRNA, othersignals, e.g., from the human growth hormone or globin genes, can beused as well. Stable cell lines carrying a gene of interest integratedinto the chromosomes can also be selected upon co-transfection with aselectable marker, such as gpt, G418 or hygromycin. It is advantageousto use more than one selectable marker in the beginning, e.g., G418 plusmethotrexate. The plasmid pC4 is digested with restriction enzymes andthen dephosphorylated using calf intestinal phosphatase by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete IL-12 antibody is usedcorresponding to HC and LC CDR regions of an IL-12 antibody of thepresent invention, respectively, according to known method steps.Isolated nucleic acid encoding a suitable human constant region (i.e.,HC and LC regions) is also used in this construct.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 micrograms of the expression plasmid pC4 iscotransfected with 0.5 micrograms of the plasmid pSV2-neo usinglipofectin. The plasmid pSV2neo contains a dominant selectable marker,the neo gene from Tn5 encoding an enzyme that confers resistance to agroup of antibiotics including G418. The cells are seeded in alpha minusMEM supplemented with 1 microgram/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexateplus 1 microgram/ml G418. After about 10-14 days, single clones aretrypsinized and then seeded in 6-well petri dishes or 10 ml flasks usingdifferent concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM,800 nM). Clones growing at the highest concentrations of methotrexateare then transferred to new 6-well plates containing even higherconcentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). Thesame procedure is repeated until clones are obtained that grow at aconcentration of 100-200 mM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reverse phaseHPLC analysis.

Example 2 Comparison of the Therapeutic Efficacy of Anti-IL-12p35 andAnti-IL-12/23p40 Antibodies in Murine Experimental AutoimmuneEncephalomyelitis (EAE)

Summary

This set of studies was performed to investigate the therapeuticefficacy of IL-12 or IL-12/IL-23 specific neutralization in a mousemodel for multiple sclerosis, experimental autoimmune encephalomyelitis(EAE). Neutralizing rat anti-mouse monoclonal antibodies (mAbs) specificfor the p35 subunit of IL-12 or the p40 subunit that is shared betweenIL-12 and IL-23 were administered either prior to disease induction,prior to disease onset, or after disease was ongoing. In all cases, onlyanti-p40 demonstrated therapeutic potential. These data suggest thatIL-23 is the predominant contributor to disease pathogenesis in thisautoimmune model.

Abbreviations:

-   -   IL Interleukin    -   mAb Monoclonal antibody    -   EAE Experimental autoimmune encephalomyelitis    -   Th T helper cell    -   IFNγ Interferon gamma    -   cs Clinical score    -   MBP Myelin basic protein    -   PK Pharmacokinetics        Introduction

Biologically active IL-12 exists as a heterodimer comprised of 2covalently linked subunits of 35 (p35) and 40 (p40) kilo Daltons.Several lines of evidence have demonstrated that IL-12 can induce robustTh1 immune responses that are characterized by production of IFNγ andIL-2 from CD4⁺ T cells. Inappropriate Th1 responses, and thus IL-12expression, are believed to correlate with many immune-mediateddiseases, such as multiple sclerosis, rheumatoid arthritis, inflammatorybowel disease, insulin-dependent diabetes mellitus, and uveitis. Inanimal models, IL-12 neutralization was shown to ameliorateimmune-mediated disease. However, these studies neutralized IL-12through its p40 subunit. The recent description of IL-23 (1), aheterodimeric cytokine that shares the p40 subunit, made it important todetermine whether previous findings were due to IL-12 or IL-23 activity.Therefore, p35 and p40 specific neutralization were compared in a mousemodel of autoimmunity, experimental autoimmune encephalomyelitis (EAE).Neutralizing antibodies specific for IL-12p35 had no effect on EAEprogression. In contrast, neutralization of both IL-12 and IL-23 with ananti-p40 mAb suppressed clinical signs of EAE, whether antibody wasadministered before or after Th1 differentiation. This data suggeststhat the activity of anti-p40 treatment in EAE is based solely onneutralization of IL-23.

Methods and Materials

Mice

Female C3H/HEB/FEJ mice (Jackson Laboratories, Bar Harbor, Me.) wereused in pharmacokinetic analyses. For EAE studies, female B10.PL(H-2^(n)) mice were purchased from the Jackson Laboratories, and wereused between 6-8 weeks of age. All animals were maintained according toIACUC guidelines under approved protocols.

Antibodies

C17.8 (rat anti-mouse IL-12/IL-23p40, IgG2a), and C18.2 (rat anti mouseIL-12p35, IgG2a) hybridomas provided by Dr. Giorgio Trinchieri and theWistar Institute (Philadelphia, Pa.). Ascites was generated at HarlanBioproducts (Indianapolis, Ind.) and purified by protein G affinity.

Serum PK of Rat Anti-Mouse Antibodies

Female C3H/HEB/FEJ mice, approximately 20-25 grams, were individuallyweighed and treated with a single 5 mg/kg intraperitoneal dose of ¹²⁵Ilabeled antibody (C17.8, C18.2), with a constant dose volume/mouse of 10mL/kg. Retro-orbital bleeds were taken from anesthetized mice at 30minutes, 6 and 24 hours, 4, 7, 11 and 18 days. Blood samples wereallowed to stand at room temperature for at least 30 minutes, but nolonger than 1 hour, and were then centrifuged at approximately2,500-3,500 rpm for 10-15 minutes. Approximately 50 uL aliquots of eachserum sample were counted for ¹²⁵I using a LKB Compugamma 1282 counter(Wallac, Gaithersburg, Md.). 10 mL aliquots of the injectates were alsocounted. The average fraction of injected counts at each time point wascalculated and multiplied by the total mg of antibody injected todetermine the total mg remaining in the serum at each time point. Datais shown as the mean mg of mAb in the sera +/−s.d. with 5-10 animals ineach group.

EAE Induction and Scoring

For EAE induction, female B10.PL mice were injected subcutaneously overfour sites on the back with a total of 100 μl of CFA (containing 200 μgMycobacterium tuberculosis Jamaica strain) combined with 200 μg guineapig-MBP (Sigma). Mice also received 200 ng pertussis toxin (ListBiological, Campbell, Calif.) i.p. in 0.2 ml PBS at the time ofimmunization and 48 hours later. Mice received i.p. injections of C17.8(anti-IL-12p40) or C18.2 (anti-IL-12p35) monoclonal antibodies dilutedto 100 mg/kg (C18.2) or 20 mg/kg (C17.8) in PBS, on indicated days.Control mice received PBS or Rat IgG (Biosource) at 20 mg/kg in PBS.

Animals that demonstrated clinical signs (cs) were scored as follows:limp tail or waddling gait with tail tonicity 1, waddling gait with limptail (ataxia) 2, ataxia with partial limb paralysis 2.5, full paralysisof one limb 3, full paralysis of one limb with partial paralysis ofsecond limb 3.5, full paralysis of two limbs 4, moribund 4.5, death 5.Animals that scored a 5 were not included in the mean daily cs analysisfor the rest of the experiment. Daily cs are averaged for the group, andmean incidence, day of onset, highest acute cs, cumulative cs, cs/day,number of relapses and relapse severity±sem are described. Meancumulative cs per group was calculated by averaging the sum of dailyclinical scores for individual animals. Cs/day was calculated bydividing the cumulative cs by the number of days the animal remained inthe study. To determine the mean day of onset, animals not developingEAE were not included in the analysis. To determine the mean highest cs,mice not developing EAE were assigned a value of “0” and included in theanalysis. Relapses were defined by a full point drop in clinical scoresustained for at least 2 observed days followed by a full point increasein clinical score sustained for at least 2 observed days.

Results and Discussion

Anti-p35 and Anti-p40 Antibodies have Identical Pharmacokinetics

To establish the clearance rates of anti-p40 and anti-p35 antibodies,normal mice were injected with a single 5 mg/kg dose of ¹²⁵I labeledantibodies and circulating levels were measured for 11 days postantibody administration. Anti-p35 and anti-p40 had overlappingpharmacokinetics, demonstrating that clearance rates are identical innormal mice (2). The expected clearance rate of each mAb isapproximately 7-10 days. Although this is a single dose PK study, thesedata support once weekly dosing for in vivo studies.

Only Anti-p40 Treatment Prior to EAE Induction is Protective

To determine the relative roles of IL-12 and IL-23 in immune-mediateddiseases, we utilized a murine model for multiple sclerosis, relapsingexperimental autoimmune encephalomyelitis (EAE). Upon EAE induction withmyelin basic protein (MBP) in adjuvant, B10.PL mice typically exhibit aninitial episode of paralysis (acute disease), then recover eitherpartially or completely and progress through multiple relapses and/orchronic EAE. It has long been assumed that EAE is dependent upon IL-12expression since IL-12 is believed to be a primary mediator of Th0 toTh1 differentiation. However, to distinguish the potential role of IL-23in EAE induction, neutralizing concentrations of anti-p40 (IL-12 andIL-23) or anti-p35 (IL-12 only) antibodies were established one dayprior to immunization for EAE (Day −1). Onset of disease can varybetween animals; therefore, treatment was repeated 7 and 14 days laterto ensure that anti-p35 and IL-p40 antibodies were present during Th1differentiation. Several in vitro neutralization studies havedemonstrated that the anti-40 mAb is 5 times more effective inneutralizing IL-12 than the anti-p35 mAb (data not shown). Therefore,the dose of anti-p35 mAb was adjusted to be 5 fold higher than anti-p40in all EAE experiments. In two separate experiments, mice treated withRat IgG isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg) didnot demonstrate protection from disease. It is important to note thatperipheral administration of a non-specific control antibody (Rat IgG)did not alter the clinical course of disease when compared tonon-treated mice with EAE. In both studies, mice treated with anti-p40mAb (20 mg/kg) exhibited nearly complete inhibition of EAE clinicalsigns. Remarkably, suppression of disease extended beyond the expectedrate of antibody clearance through 70 days post EAE induction. In eachexperiment, only one animal treated with anti-p40 exhibited twoconsecutive days of EAE clinical signs, and each demonstrated a lateonset and significantly lower acute clinical scores, cumulative clinicalscores, and no relapses in disease (Table 1). These results demonstratethat neutralization of IL-12 and IL-23 through the shared p40 subunitprovided nearly complete protection from EAE. In contrast, specificneutralization of IL-12 only via anti-p35 was ineffective. These datastrongly suggest that EAE is not mediated by IL-12.

Only Anti-p40 Treatment Just Prior to Disease Onset is Protective

Although prophylactic treatment completely protected mice from EAE, itremained to be determined if IL-12 specific neutralization would beprotective once the Th1 population was established in vivo. Therefore,in a separate set of experiments, mice were treated with either acontrol antibody (Rat IgG), anti-p35, or anti-p40 monoclonal antibodiesten days after EAE induction, but prior to disease onset. Since typicalimmune responses occur within 7 days, this time point should reflect theeffects of anti-IL-12 or anti-IL-23 mAbs on differentiated Th1 cells.EAE onset can vary between animals, therefore treatment was repeated 7and 14 days later to ensure that anti-p35 and anti-p40 antibodies werepresent during the onset of disease. In two separate experiments, micetreated with isotype control antibody (20 mg/kg) or anti-p35 (100 mg/kg)were not protected from disease, when compared to untreated EAE mice.However, mice treated with anti-p40 mAb (20 mg/kg) were significantlyprotected from EAE. As shown in the previously described studies,disease suppression was observed well beyond the time required forclearance of peripherally administered antibody through day 70 post EAEinduction. Considering that antibody was not administered until afterTh1 differentiation (day 10), it was not surprising that diseaseincidence, day of onset, and the highest clinical score during acute EAEwere not different in any group (Table 2). However, in both experiments,mice receiving anti-p40 exhibited significantly lower cumulativeclinical scores, clinical scores per day, and relapse severity.

Only Anti-p40 Treatment During Established EAE is Protective

The most difficult, but clinically relevant, hurdle for any therapy isto suppress established disease. Therefore another set of experimentswas performed in which mice were immunized for EAE, then divided intotreatment groups once disease was ongoing. Approximately 30 days postEAE induction, mice had progressed through the acute phase of disease.At this time, animals were divided into groups with comparablecumulative and daily clinical scores. Treatment was repeated 7 and 14days later to ensure that antibodies were available in neutralizingconcentrations during the transition from acute to chronic orremitting-relapsing disease. Only anti-p40 treatment (20 mg/kg)ameliorated disease when compared to either isotype control antibody (20mg/kg) or anti-p35 (100 mg/kg) treated animals. Disease suppression wasobserved through day 80 post EAE induction. In both experiments,analysis from the first day of treatment through day 80 demonstratedthat mice receiving anti-p40 exhibited lower cumulative clinical scores,clinical scores per day, and the least highest clinical score posttreatment. These data suggest that not only is IL-23 likely to mediateTh1 differentiation (Table 1) and EAE induction (Table 2), but IL-23also contributes to the effector phase of chronic immune-mediated (e.g.,autoimmune) responses (Table 3). Therefore, anti-p40 treatment can offertherapy at any time in the progression of immune-mediated diseases.

Conclusions

The understanding of the role of IL-12 in immune function has been basedon studies of the p40 subunit of IL-12. Therefore, a side-by-sidecomparison of neutralization of the IL-12 specific p35 subunit versusthe p40 subunit shared between IL-12 and IL-23 was conducted in ananimal model of autoimmune disease. Neutralization via anti-p40significantly inhibited EAE when mAb was administered at any time point.However, IL-12 specific neutralization was completely ineffective.Therefore, our data shows that IL-12 does not contribute to thisautoimmune model and that IL-23 is expected be the more prominentmediator of autoimmune T cell responses.

Example 3 p40 Neutralizing Epitope

Summary

The epitope for a neutralizing antibody (p40 mAb) against human p40subunit of IL-12 and IL-23 was determined based on the crystal structureof the Fab/IL-12 complex. The epitope is located on the D1 domain(residues 1-88) of the p40 subunit of human IL-12. This region isdistant from the p40/p35 interface and is expected to be also availableon the p40 subunit of IL-23. The residues involved in antigen-antibodybinding are discontinuous (Table 4) and comprise a unique conformationalepitope. Antibodies against this epitope or portions of it andneighboring regions will lead to blockage of IL-12 and IL-23 functionsmediated through this part of p40 subunit.

Introduction

A fully human monoclonal antibody (p40 mAb) directed against human IL-12has been shown to be a potent neutralizer of IL-12 and IL-23 function.It has been shown that the p40 mAb binds to the p40 subunit and blocksthe binding of both cytokines to their receptors. Because the p40subunit is shared between IL-12 and IL-23, the detailed interactionsbetween IL-12 and the p40 mAb define an important common neutralizingepitope, which may in turn shed some light on the cytokine-receptorinteractions.

Epitope Determination Based on p40 Fab/IL-12 Crystal Structure

The p40 mAb was produced from a mammalian cell line in culture andpurified by protein A column. The p40 mAb (70 mg) was digested withpapain (0.25 units of papain per milligram of IgG) in activation buffer(0.03M sodium phosphate, 0.15M NaCl, 0.01M EDTA, 0.0072M L-cysteine, pH7.0) at 37° C. for 2 hours. Digestion was monitored by Surface-EnhancedLaser desorption ionization (SELDI) mass spectrometry. Iodoacetamide(0.5M) was used to stop the digestion. Fc was removed by immobilizedprotein G. The p40 Fab was further purified by gel filtration on aSuperdex 200 16/60 column. A total of 44 mg purified p40 Fab wasobtained and its purity analyzed by SDS-PAGE.

Recombinant human IL-12 was produced in culture from a stablytransfected cell line over-expressing the p40 and p35 subunits andpurified with a p40 mAb affinity column. The protein fractions werecollected and dialyzed into 10 mM Tris, 100 mM NaCl, pH 7.4 andconcentrated to 2.5 mg/ml. IL-12 was deglycosylated by incubating withseveral combinations of deglycosylation enzymes (PNGase F, Sialadase,Endo-O-glycosydase, α-, β-galactosidase, α-mannosidase, fucosidase[about 5 mU-10 U/100 ug protein]) for 3 days at 37° C. under argon.

Deglycosylated IL-12 was mixed with an excess of the p40 Fab. TheIL-12/p40 Fab complex was purified by size exclusion chromatography in10 mM Tris, 50 mM NaCl, pH 7.4. The isolated complex was concentrated toapproximately 4 mg/ml. The IL-12/p40 Fab complex was crystallized usingthe sitting drop vapor diffusion method by combining the above proteincomplex solution in 1:1 volume ratio with a reservoir solution of 50 mMTris, pH 7.0, 16% PEG 3350. Cubic, pyramidal or rod shaped crystals oftypically 50-150 μm in size appear within two weeks at 16° C.

The crystals were harvested, and soaked in the mother liquor plus 30%ethylene glycol and flash frozen in liquid nitrogen for X-ray datacollection. The best data set was collected (360 degrees, 0.5degree/frame, 10 second exposure per frame) to a diffraction limit of2.8 Å at Advanced Photon Source (APS), Argonne National Laboratory,(Axas-ComCat). The diffraction data were processed with Denzo andScalePack. (Otwinowski & Minor, Methods Enzymol. 276: 307-326) The spacegroup for this crystal form is P2₁2₁2₁ with cell dimensions of a=116.8Å, b=55.77 Å, c=182.96 Å, and α=β=γ=90°. There are 27,141 independentreflections and the data was ˜90% complete at 3.0 Å (I/sigma=5.2,R_(sym)=9%.).

The crystal structure was solved by molecular replacement as implementedin CNX (Accelrys, CA). The search models were the published IL-12structure (PDB code 1F45) for IL-12 and a homology model for p40 Fabbased upon a Fab crystal structure (PDB code 1VGE). The molecular modelsfor IL-12 and the p40 Fab were visually inspected and manually adjustedusing XtalView. The structure refinement was carried out with the CNX.The molecular models were validated with InsightII (Accelrys, CA). Themolecular structure of the bound complex of IL-12/p40 Fab is shown inFIG. 1 in a ribbon representation.

The p40 mAb binding site (p40 epitope) on the moleuclar surface insurface and ribbons representations is shown in FIG. 2. The bindingepitope residues are defined as any surface exposed p40 residues(relative solvent accessibility of 0.1 or greater) with any atoms within4 Å of any atoms of the antibody, according to a generally acceptedconvention. The surface accessibilities were calculated by ICM (Molsoft,CA) using the default parameters. Residues comprising the p40 mAbbinding epitope on IL-12 p40 are listed in Table 4, along with theirsurface exposed areas (sf) and relative accessibilities (sf ratio). (Thedefinition of exposed surface area of an amino acid residue within thecontext of protein has been well accepted in the field. In particular, awater molecule with a radius of 1.4 A is rolled along the surface of aprotein and the amount of area obtained by this calculation for aspecific residue is assigned to the expoed surface area of that residue.The total surface area for an amino acid in fully extended conformationis also available. The surface accessibility (sf ratio) is then theratio of the exposed surface area over the standard surface area. Thesetwo values together give us ideas whether a residue is exposed on theprotein surface. Apparently, only surface exposed residues can beinvolved in binding other molecules such as antibody. However, we do notneed to explain all of this in the text. JL.)

It is clear from Table 4 and FIG. 2 (right panel) that the binding siteon p40 is discontinuous and constitutes a large number of surfaceexposed residues that are distributed over an irregular surface. Theantibody-antigen interaction buries a total of 1758 Å² of accessiblesurface on IL-12 and p40 mAb. The interactions appear to be dominated bythree salt bridges: R59(H)-E59(p40), R98(H)-E45(p40) andR99(H)-D62(p40). Also, there are contributions to the antibody-antigeninteraction that are due to hydrophobic or Van der Waals forces.

The residues on the IL-12p40 antibody involved in IL-12 bindingidentified based upon the co-crystal structure herein are shown in Table5 below. All surface exposed residues of the anti-p40 antibody with anyatoms within 4 A of the p40 subunit are considered part of these bindingresidues. Conservative changes to any one or more of these residues mayproduce mutant antibodies that are of similar potency. Examples of suchconservative substitutions include, without limitation, R59K, R98K andR99K in VH (e.g., SEQ ID NO:7) and DIE in VL (e.g., SEQ ID NO:8).

Also, saturation mutagenesis at each position shown in Table 5 can beperformed (e.g., changing the wild type (WT) amino acid sequence to anyother amino acid, possibly with the exception of Cysteine) to identifymutations causing the resulting antibody to have increased, decreased orsubstantially similar activity (e.g., binding). Binding of the resultingantibody to the p40 subunit can be tested pursuant to any suitablebinding assay. Saturation mutagenesis can be used, for example, tocreate a more or less potent antibody or an equally potent antibodyhaving different properties, i.e., properties other than potencyresulting from a change in the variable region sequence (e.g., size orother structural changes to the variable region).

The saturation mutagenesis can also be performed using more than onesubstitution, for example, two, three, or more positions for eachexperiment. This can be done as individual clones or libraries followedby selection or screening in an approriate format, such as phagedisplay. Furthermore, individual or combination mutations at thepositions in Table 5 that are desirable to regulate activity can becombined to create additional combination mutants of similar or betterpotency.

Advantage

The p40 mAb binding site is distant from the p40/p35 association site(FIG. 1). The p19 subunit of IL-23 is evolutionarily related to p35 ofIL-12 with significant sequence homology. It is likely that p19associates with p40 in a fashion similar to p35. Therefore, the p40 mAbbinding region is also distant from the p40/p19 interaction in IL-23.The epitope identified in the present invention is equally available inboth IL-12 and IL-23; accordingly, it is not surprising that p40 mAb canactively block the functions of both cytokines.

IL-12 (p40/p35) and IL-23 (p40/p19) interact with their respectivereceptors (IL-12Rβ1/β2 and IL-12Rβ1/IL-23R) in a similar fashion. Theyinduce similar signaling cascades. The details of the cytokine-receptorinteractions, however, are not clearly defined at the molecular level.The newly defined p40 mAb epitope could represent a biologicallyimportant site for the interactions between the IL-12 family ofcytokines with their shared receptor, IL-12Rβ1. Therefore, the epitopeis an important target for the therapeutic intervention using monoclonalantibodies, peptides, recombinant proteins, small molecules and othernatural or synthetic agents.

Example 4 Epitope Mapping of p40 mAb on IL-12p40 Using MutationalAnalysis

Summary

ELISA binding of IL-12p40 mutants with p40 mAb was carried out to verifythe binding epitope. Based on the crystal structure of p40 Fab/IL-12complex, 7 single mutants and 2 double mutants were generated. Themutated residues are located in the p40 Fab contact region in the domainI (D1) of p40 subunit. The relative binding affinity showed that threenegatively-charged residues, E45, E59 and D62, contribute significantlyto binding interactions with p40 mAb. The other residues, M23, L40 andS43, have less but appreciable contribution. This mutational analysisconfirms that p40 mAb recognizes domain I of p40 subunit and residuesE45, E59, D62, M23, L40, and S43 are part of the binding epitope.

Materials and Methods

Seven human p40 single mutants, M23T, L40T, S43R, E45A, E45R, E59R, andD62R, and two double mutants, S43R/E45A and S43R/E45R, are used in thestudies. The wide-type human p40, WT hu-p40, was used as the control.The p40 mutants were transiently expressed in HEK293E cells. Thesupernatants were used for binding assays.

In brief, MSD high bind plates (Meso Scale Discovery, MD) were coatedwith 5 μl of a capture monoclonal antibody (5 μg/ml) at room temperaturefor 1 hour. The capture monoclonal antibody recognizes p40 subunit butdoes not compete with p40 mAb. One-hundred and fifty (150) μl of 5% MSDBlocker A buffer was added to each well and incubated for 1 hr at roomtemperature. Plates were washed three times with 0.1 M HEPES buffer (pH7.4). These protein-charged ELISA plates were incubated with 25 μl ofdifferent transiently expressed p40 mutant supernatants (1:10 dilutionwith 0.1 M HEPES buffer, pH 7.4) for 1 hr at room temperature. Theplates were washed three times with 0.1 M HEPES buffer (pH 7.4).Twenty-five (25) μl of different concentrations of MSD Sulfo-TAG labeledp40 mAb, ranging from 0 to 20 μg/ml, were dispensed to micro-wells.After incubation for 2 hrs shaking at room temperature, plates werewashed 3 times with 0.1 M HEPES buffer (pH 7.4). One hundred and fifty(150) μl of diluted MSD Read Buffer T was dispensed into each well andthe plates were analyzed with a SECTOR imager (MSD).

Results

As shown in FIG. 3, E45R, E59R, and D62R each reduced the mutein p40binding affinity to p40 mAb significantly, compared to the wild typep40. E45A had significant but less dramatic effect on the bindingcompared to E45R. M23T, L40T and S43R each had moderate effect uponbinding.

The relative binding affinity of p40 mAb to different mutant proteinswas further analyzed by a capture assay. As shown in FIG. 4, E45R, E59Rand D62R each nearly completely abolished antigen binding. The othermutations, M23T, L40T and S43R, in the interface, each reduced bindingby approximately 40%. E45A reduced binding by about 70%.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

TABLE 1 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day −1 (prior to Th1 differentiation). Relapse GroupIncidence Mortality Day of onset Highest acute cs^(a) Cumul cs^(b)Cs/day No. of relapses severity P-2001-060 Rat IgG 13/13 4/13 30.5 ± 3.23.6 ± 0.3 71.4 ± 14.1 1.2 ± 0.2 1.3 ± 0.2 3.6 ± 0.2 Anti-p35 11/13 8/1329.6 ± 3.4 3.5 ± 0.5 45.5 ± 11.5 0.8 ± 0.2 1.2 ± 0.1 4.0 ± 0.3 Anti-p40 1/13 0/13 40.0 0.1 1.2 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 P-2001-079No treatment 6/7 0/7  24.7 ± 2.7 3.2 ± 0.6 110.4 ± 20.4  1.7 ± 0.3 1.0 ±0.4 3.8 ± 0.1 Rat IgG 9/9 2/9  29.1 ± 2.9 3.8 ± 0.2 90.6 ± 10.1 1.5 ±0.1 0.3 ± 0.2 4.7 ± 0.3 Anti-p35 10/10 1/10 30.0 ± 2.6 3.9 ± 0.2 94.9 ±17.8 1.4 ± 0.2 0.7 ± 0.3 3.9 ± 0.2 Anti-p40  1/10 0/10 61.0 0.3 1.6 ±1.1 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 ^(a)clinical score (cs) ^(b)cumulativecs Mice were treated as described and clinical scores were analyzed fromday 0 through 70 days post EAE induction. Data is shown as the mean pergroup ± s.e.m.

TABLE 2 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 10 (after Th1 differentiation). Highest Relapse GroupIncidence Mortality Day of onset acute cs^(a) Cumul cs^(b) Cs/day #relapses severity P-2001-037 No treatment 7/8 0/8  30.6 ± 2.7 3.2 ± 0.551.5 ± 14.4 0.8 ± 0.2 0.3 ± 0.2 3.3 ± 0.8 Rat IgG  9/10 0/10 25.9 ± 2.72.7 ± 0.5 74.7 ± 15.8 1.2 ± 0.2 0.6 ± 0.2 3.7 ± 0.4 Anti-p35  9/10 0/1025.8 ± 2.6 2.5 ± 0.4 58.8 ± 15.6 1.0 ± 0.2 0.7 ± 0.3 3.2 ± 0.3 Anti-p406/7 0/7  34.7 ± 6.3 1.6 ± 0.5 14.9 ± 7.5  0.2 ± 0.1 0.3 ± 0.2 1.5 ± 0.5P-2001-053 No treatment 8/9 2/9  15.8 ± 2.2 2.1 ± 0.6 56.4 ± 19.1 0.9 ±0.3 0.6 ± 0.3 3.3 ± 0.5 Rat IgG  9/10 4/10 20.0 ± 2.5 3.8 ± 0.5 70.1 ±17.7 1.3 ± 0.2 0.3 ± 0.2 4.2 ± 0.4 Anti-p35 10/10 1/10 16.5 ± 1.1 3.2 ±0.3 93.8 ± 15.7 1.4 ± 0.2 0.8 ± 0.2 3.2 ± 0.3 Anti-p40 10/10 2/10 13.6 ±1.1 2.7 ± 0.5 23.2 ± 7.9  0.4 ± 0.1 0.4 ± 0.3 2.0 ± 0.4 ^(a)clinicalscore (cs) ^(b)cumulative cs Mice were treated on days 10, 17, and 24and clinical scores were analyzed from day 0 through 70 days post EAEinduction. Data is shown as the mean per group ± s.e.m.

TABLE 3 EAE clinical scores with IL-12 and IL-23 neutralizationinitiated on day 30 (during established EAE). From first treatmentthrough 80 days post EAE induction Group Pre-Tx^(a) Mortality Cumulcs^(b) Cs/day Highest cs Lowest cs # relapses Relapse severity P-2002-01No treatment 2.7 ± 0.6 1/5 132.9 ± 29.3  3.3 ± 0.3 4.1 ± 0.2 2.4 ± 0.50.6 ± 0.4 3.7 ± 0.0 Anti-p35 2.3 ± 0.7 1/5 135.9 ± 16.5  2.7 ± 0.3 3.8 ±0.4 1.8 ± 0.3 2.0 ± 0.4 3.7 ± 0.3 Anti-p40 2.0 ± 0.2 1/6 75.6 ± 16.1 1.9± 0.3 2.8 ± 0.5 1.0 ± 0.4 0.7 ± 0.3 2.5 ± 1.0 P-2002-093 Rat IgG 1.7 ±0.8 1/5 87.7 ± 16.4 2.1 ± 0.2 3.7 ± 0.4 1.2 ± 0.5 1.5 ± 0.5 3.8 ± 1.0Anti-p35 1.9 ± 0.7 1/5 98.2 ± 9.7  2.2 ± 0.1 3.7 ± 0.4 1.4 ± 0.4 1.5 ±0.3 3.3 ± 0.2 Anti-p40 2.4 ± 0.8 0/5 71.7 ± 21.6 1.5 ± 0.4 2.9 ± 0.6 0.8± 0.5 1.3 ± 0.3 2.7 ± 0.6 ^(a)mean clinical score per group on the firstday of treatment (Tx) ^(b)clinical score (cs)

TABLE 4 Residues contributing to the p40 mAb binding epitope onIL-12/p40 Number Residue sf Sf Ratio 15 Trp 94.2 0.36 17 Pro 69.3 0.4618 Asp 132.9 0.86 19 Ala 27.7 0.22 20 Pro 131.5 0.88 21 Gly 18.7 0.21 23Met 142.4 0.66 40 Leu 32.7 0.16 41 Asp 69 0.45 42 Gln 145.8 0.77 43 Ser58.2 0.46 45 Glu 139.2 0.74 46 Val 95.8 0.57 47 Leu 95 0.48 54 Thr 77.20.51 55 Ile 32.7 0.17 56 Gln 111 0.58 58 Lys 69.2 0.32 59 Glu 83.2 0.4460 Phe 113.3 0.51 61 Gly 68.2 0.77 62 Asp 41.3 0.27

TABLE 5 Residues on p40 mAb involved in binding to IL-12/p40 epitopeHeavy chain Light chain residues residues S28 D1 T31 S30 Y32 W32 W33 Y49D57 S56 R59 N92 R98 I93 R99 Y94 R100 P101 G102 Q103

What is claimed is:
 1. A method for treating psoriatic arthritis in ananimal, comprising administering a composition comprising an effectiveamount of an isolated anti-IL-12 antibody, wherein said antibody bindsto a conformational epitope of IL-12 protein comprising residues 15,17-21, 23, 40-43, 45-47, 54-56 and 58-62 of the amino acid sequence ofSEQ ID NO:9, to said animal.
 2. The method according to claim 1, whereinsaid effective amount is 0.001-50 mg/kilogram of said animal.
 3. Themethod according to claim 1, wherein said administering is by at leastone mode selected from parenteral, subcutaneous, intramuscular,intravenous, intrarticular, intracapsular, intracartilaginous,intracavitary, intraosteal, intrapelvic, intraperitoneal, intraspinal,intrasynovial, buccal, sublingual, intranasal, and transdermal.
 4. Themethod according to claim 1, further comprising administering at leastone composition comprising an effective amount of at least one compoundor protein selected from the group consisting of a detectable label orreporter, a TNF antagonist, an antirheumatic, a muscle relaxant, anarcotic, a non-steroid anti-inflammatory drug (NSAID), an analgesic, ananesthetic, a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, a betaagonist, an inhaled steroid, an epinephrine or analog, a cytokine, and acytokine antagonist.
 5. A method for treating psoriatic arthritis in ananimal, comprising administering a composition comprising an effectiveamount of an isolated anti-IL-12 antibody, wherein said antibody bindsto a conformational epitope of IL-12 protein comprising residues 23, 40,43, 45, 59, and 62 of the amino acid sequence of SEQ ID NO:9, to saidanimal.
 6. The method according to claim 5, wherein said effectiveamount is 0.001-50 mg/kilogram of said animal.
 7. The method accordingto claim 5, wherein said administering is by at least one mode selectedfrom parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intracapsular, intracartilaginous, intracavitary,intraosteal, intrapelvic, intraperitoneal, intraspinal, intrasynovial,buccal, sublingual, intranasal, and transdermal.
 8. The method accordingto claim 5, further comprising administering-at least one compositioncomprising an effective amount of at least one compound or proteinselected from the group consisting of a detectable label or reporter, aTNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, anon-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic,a sedative, a local anesthetic, a neuromuscular blocker, anantimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, a betaagonist, an inhaled steroid, an epinephrine or analog, a cytokine, and acytokine antagonist.